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Act I.

In the garden of the Cossack Kochubey, overlooking the River Dnepr (Scene 1), Kochubey's daughter Mariya remains behind while the other girls sail down the river. She loves the elderly hetman Mazepa, but the cossack Andrey is in love with her. The peasants dance a gopak to honour Mazepa. Mazepa asks Mariya's father for her hand in marriage, but Kochubey says that Mazepa is too old for her and refuses his consent. After an argument, Mariya again declares her love for Mazepa and she leaves with him. Inside Kochubey's house (Scene 2), Kochubey sends Andrey to Moscow, to denounce Mazepa to the Tsar for conspiring with the Swedes.

Act II.

In a dank dungeon beneath a palace at Belaya Tserkov, Kochubey is chained to a pillar (Scene 1). The Tsar does not believe Kochubey's accusations against Mazepa and delivers him and his comrades into Mazepa's hands. Orlik, Mazepa's henchman, tortures Kochubey, and demands to know where he keeps his treasure. Later, in a room in Mazepa's castle (Scene 2), Orlik tells Mazepa that Kochubey has not revealed his secret under torture. Mazepa decides that Kochubey must die the next day. Mariya enters, unaware of her father's fate, and Mazepa tells her of his plans to rule over an independent Ukraine. After they reaffirm their love, Mazepa leaves. Mariya's mother Lyubov appears, and tells her that Kochubey is to be executed. The following morning, in a field with a scaffold (Scene 3), a crowd has gathered to witness the execution. Mazepa watches on horseback as Kochubey and Iskra are led to the scaffold. Mariya and her mother arrive just as the axes fall.

Act III opens with a symphonic tableau depicting the Battle of Poltava.

Mazepa's forces are defeated. In Kochubey's garden, now neglected and overgrown, Andrey and other Russian soldiers are pursuing fleeing Swedish soldiers. Andrey hides when he hears Mazepa and Orlik approaching. Andrey attacks Mazepa and is mortally wounded. Mazepa discovers Mariya wandering about in a daze, but he is forced to flee without her. Andrey dies in Mariya arms as she sings him a lullaby