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ACT I. In the garden of Kochubei’s farmstead, on the banks of the Dnieper, peasant girls pass by in boats, inviting Maria, Kochubei’s daughter, to join their fortune-telling games. She demurs; her father is entertaining an important guest — the Cossack chieftain Mazeppa, ruler of the Ukraine. When they are gone, she reflects that girlish pursuits hold no charm for her since the elderly Mazeppa has bewitched her heart. Andrei, overhearing her passionate declaration, offers comfort and understanding, but when she thanks him for his friendship, he replies that it is not friendship but love that inspires his sympathy. She asks his forgiveness, and they lament the fatal passions that rule them both. Bidding her a tearful farewell, Andrei rushes away.

In Kochubei’s house, the assembly hails the guest of honor. After a group of folk dancers regales them with a Hopak, Mazeppa draws his host aside to ask for his daughter’s hand. When Kochubei, appalled, points out the gulf between their ages, Mazeppa replies that the passions of a mature heart burn slowly but steadily, assuring Kochubei that Maria returns his love. The proud father refuses his consent and orders Mazeppa to leave his house. Mazeppa, affronted, calls his in guard and demands submission, but Kochubei defies him, backed by Andrei and the guests, who condemn Mazeppa’s sinful desires. Maria, torn between love and duty, throws herself between her beloved and her father; Mazeppa, preparing to leave, forces Maria to choose between them. After an agonizing moment, she runs into his arms. With his guard holding back the horrified company, he leads her away.

A group of women comforts Lyubov as she laments the loss of her child. Sending them away, she urges Kochubei to wreak vengeance on Mazeppa. Kochubei reveals that in an unguarded moment Mazeppa has let slip hints of a treasonous plan to align himself with the Swedes to overthrow the Tsar. Kochubei plots to betray Mazeppa to Peter the Great. Andrei, saying his life is of no value without Maria, volunteers to brave the Tsar’s disbelief by denouncing his trusted hetman. Kochubei and Lyubov exult that their enemy will die at the hands of executioners.

ACT II. Kochubei, imprisoned and awaiting execution, laments that the Tsar has taken Mazeppa’s slanderous word over his loyal one and turned him over to his enemy; Kochubei, who has made a false confession under torture, regrets the loss of his honor more than his life. When Mazeppa’s henchman Orlik enters, demanding to know where Kochubei has hidden his treasure, Kochubei replies that, since Mazeppa has stolen his and his daughter’s honor, the only treasure left him is vengeance, which rests with God. As Orlik summons the torturers again, Kochubei falls senseless.

In a room in his palace, Mazeppa resolves to kill Kochubei and Andrei, but his soul is troubled by guilty visions, and he wonders how Maria will react when she learns of her father’s fate. Orlik comes to report Kochubei’s refusal to cooperate, and Mazeppa sends him to prepare for the execution. Alone, he reflects on the rejuvenating effects of Maria’s love. She enters to complain of his recent neglect, remaining unconvinced by his avowals that he loves her more than glory or power. To assuage her fears, he confides his revolutionary plans, saying he may soon ascend the throne of an independent Ukraine. Maria is swept away by visions of his glory; when he warns that failure will mean the gallows, she vows to die with him and reaffirms her love. When Mazeppa asks whether he is dearer to her than her father, Maria, perturbed, assures him of her devotion; they embrace, asking one another’s forgiveness. Left alone, Maria envisions her parents in distress. Lyubov enters unexpectedly and implores her to save her father’s life. Maria, at first uncomprehending, finally realizes what has happened and, overcome by guilt, falls in a faint. Reviving quickly, she hurries away with Lyubov to plead for Kochubei’s life.

In a field near a scaffold, an unruly mob awaits the execution. A drunken Cossack sings a song, berated by the crowd, which bows subserviently to Mazeppa when he rides by. Kochubei and Iskra are brought in. They kneel in repentant prayer, then mount the scaffold. As the crowd surges forward, Maria and Lyubov rush in, only to see the axes fall. Maria collapses in her mother’s arms.

ACT III. In the ruins of Kochubei’s farmstead, now a battlefield, Andrei, thirsting for vengeance, regrets that he has pursued Mazeppa in vain. Surveying the place where his childhood love for Maria blossomed, he laments his lost dream of happiness, wishing for oblivion and peace. He withdraws as Mazeppa and Orlik ride in, fleeing the victorious Russians. When Orlik leads the horses off, Mazeppa, now branded a traitor, bemoans the downturn in his fortunes. Andrei comes forward to berate him for destroying the happy home that once stood on the battleground and challenges him to a duel to the death. Mazeppa replies that his grey hairs and his misfortunes should command respect, but when Andrei, unmoved, rushes at the old man with his sabre, Mazeppa draws his pistol and shoots. Andrei falls wounded. Mazeppa, calling for Orlik, is stopped by the appearance of Maria, mad with grief over her father’s death. She fails to recognize Mazeppa, who tries briefly to bring her to her senses before abandoning her at Orlik’s urging to save himself. Alone, Maria comes upon the wounded Andrei. Imagining him to be a sleeping child, she cradles him in her arms and sings him a lullaby. Andrei, coming to, tries in vain to penetrate her delirium, but his eyes grow dim, and he dies with a last farewell on his lips, as Maria gently rocks him in her lap.