Verdi's opera is based on a French play called Le Pasteur — The Pastor. The setting is a devoutly religious community in Austria. After the overture, ACT ONE begins as the preacher Stiffelio is expected to return after a long mission abroad. People are waiting for him in the home of Stankar, the father of Stiffelio's wife, Lina. The opera's pious atmosphere is established right from the start, as the old preacher Jorg takes up a bible and offers a fervent prayer for the success of Stiffelio's holy cause.
Stiffelio arrives, and as the people greet him, he tells them an odd story he heard from the boatman who brought him home. The boatman said that about a week ago, he saw a man and a woman at a window, in a house near the river, very early in the morning. They seemed nervous, and the man left the house by jumping from the window into the water, dropping a sheaf of papers in the process. The boatman retrieved the papers, and suspecting some sort of skullduggery, he gave them to Stiffelio, the village's spiritual leader.
Now, Stiffelio takes the papers from his coat, and everyone asks what he plans to do with them. Stiffelio says that to return them, he would have to read them first. He cites the bible as saying men should have mercy toward their brothers. So, in a spirit of forgiveness that's woven throughout opera, Stiffelio goes to the fireplace, and tosses the papers — along with any humiliating secrets they might contain — into the flames.
When all the visitors leave, Stiffelio is finally alone with Lina, and she's obviously upset about something. Stiffelio tells her about his journey and all the moral depravity he saw along the way — faithlessness and immorality — and his stories clearly disturb Lina. At first he's sympathetic, saying what a relief it is to be home with his faithful wife. But then, Stiffelio notices that Lina isn't wearing her wedding ring. He demands to know where it is but Lina doesn't answer.
As Stiffelio grows suspicious, they're interrupted by Stankar, who says Stiffelio's friends are waiting for him. The two men leave, with Stiffelio telling Lina that he'll be back soon.
When she's alone, Lina decides to write Stiffelio a letter of confession. But she's barely begun it when Stankar returns unexpectedly. He grabs the letter, and just from reading the first few lines he realizes that Lina has been having an affair with a young man named Raffaele. First, he berates his daughter for her shameful behavior. Then he forbids her from revealing the affair to anyone — especially Stiffelio — saying they must protect their family honor.
After they leave the room, Raffaele enters. Lina has apparently refused to meet with him. So he writes her a letter, which he folds and puts into a large book — a copy of the religious epic Messiah, by Klopstock. The book has a lock, and only he and Lina have keys for it.
Then, just after he puts it back on the shelf, Lina's cousin Federico shows up. Without knowing what's going on, he says he needs the book, and he tucks it under his arm and leaves. And that's not Raffaele's only problem. Jorg has been watching the whole time, and he now believes Federico and Raffaele are conspiring to smuggle letters to an illicit lover — though he doesn't know who it is.
In the next scene, everyone has gathered in a large hall for a celebration. Aside, Jorg tells a flabbergasted Stiffelio about what he's seen, and his suspicions about Federico and Raffaele. Then someone asks Stiffelio what he intends to preach about in his next service. He says the sermon will be about deception and betrayal — a message taken from Klopstock's "Messiah." He takes the book from Federico. Stiffelio finds the volume locked, and Lina's cousin Dorotea innocently blurts out that Lina has a key. When Lina refuses to unlock the book, Stiffelio tears it open himself. He quickly finds the letter, but before he can read it Stankar grabs it from him and rips it to shreds.
In the final ensemble, Stiffelio flies into rage. And aside, Stankar and Raffaele arrange to meet later that night, in the graveyard, to fight a duel.
ACT TWO begins with a dark, orchestral introduction. We see Lina visiting her mother's tomb. It's in the same graveyard where Stankar and Raffaele have agreed to meet. Raffaele arrives alone, and immediately goes to Lina. Despite their affair, Lina is still devoted to her Stiffelio, and tells Raffaele to leave her alone. But Raffaele says he still loves her.
They're interrupted when Stankar appears, but as the two men draw their swords and begin to duel, Stiffelio arrives. He commands them to stop fighting, in the name of God. The duel is halted, but Stankar wants Raffaele to get what's coming to him — so he coldly tells Stiffelio that it was Raffaele who seduced Lina.
That news does the trick. The pious Stiffelio is furious. He draws his own sword and approaches Raffaele. But just then, a choir begins singing inside the chapel. It's a hymn about divine forgiveness. Hearing this, Stiffelio can't go through with his attack, and drops his weapon.
ACT THREE opens in a passageway in Stankar's home, with doors leading to several rooms. Stankar is pondering his situation, and he's ambivalent. He's ashamed of his daughter's infidelity, and the dishonor it could bring his family, but he's not sure how to deal with it. In a long and dramatic aria, he actually considers suicide. Eventually, Stankar decides he must act boldly to save his family's reputation and goes off.
Stiffelio and Jorg appear, on their way to the church for services, and they encounter Raffaele. Stiffelio tells Jorg to find Lina, and send her there to meet him. Then he asks Raffaele what he would do if Lina were free to be with him. When Raffaele hesitates, Stiffelio leads Raffaele into a side room and tells him to wait, and listen — saying he'll soon hear everything he needs to know about Lina's future.
Lina arrives, and in the opera's key scene, Stiffelio confronts her. At first, he says their only option is divorce. But Lina says no — that she still loves Stiffelio. She begs him to act like a man of god, not a jealous husband, and forgive her.
Stiffelio decides that she's right. It's her seducer who deserves punishment. He heads for the other room to deal with Raffaele, but Stankar has gotten there first. As Stiffelio approaches, Stankar emerges from the room with his sword dripping blood, and says he has just avenged his family honor. Raffaele is dead.
The final scene takes place in the church where Stiffelio is set to address his followers. As the worshippers finish their prayers, Stiffelio enters and approaches the pulpit. His faith has been shaken and he's unsure what message he will deliver. Stiffelio decides to open the Bible at random, and take his inspiration from whatever passage appears. His eyes fall on a New Testament story of a woman caught in adultery, and Jesus asking those without sin to cast the first stone. Then, with Lina and Stankar prostrate before the altar and seeming to await judgement, Stiffelio reads the final passage: "And the woman rose up, and was forgiven." The congregation repeats the message, and Lina is also forgiven, as the opera ends.
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