(Don Carlo, Crown Prince of Spain, has been betrothed to Elizabeth de Valois, daughter of Henry II of France, but learns that his own father, Philip II, claims her for his bride.)

Carlo seeks consolation at the cloister of the Monastery of St. Just, where the monks chant their prayers at the tomb of Charles V, Carlo’s grandfather. His friend Rodrigo, Marquis of Posa, suggests that Carlo leave for the Netherlands to cure himself of his infatuation and to protect the Flemish against the tyranny of Spain. The two men pledge friendship; King Philip and Queen Elizabeth approach the tomb, kneel briefly and proceed on their way.

In the cloister garden, Princess Eboli, the Countess of Aremberg and their ladies entertain themselves; Eboli sings a Moorish song to the accompaniment of Tebaldo’s mandolin. As the Queen enters sadly from the monastery, Rodrigo appears, hands her a letter from Carlo and tells her that the Prince longs to see her. Elizabeth agrees to receive him, and the page leads Carlo to her side. The ladies retire. Carlo begs the Queen to obtain Philip’s leave for him to go to Flanders and then declares his love in a passionate avowal. Breaking free of Carlo’s embrace, Elizabeth turns him away. No sooner has he left than Philip reenters with his suite and, finding his wife unattended, banishes the Countess of Aremberg, who should have been at the Queen’s side. Elizabeth consoles her; the ladies depart, leaving Rodrigo to plead the Flemish cause with Philip. The King suspects that Elizabeth and Carlo may have betrayed him and asks Rodrigo to watch the lovers, warning him of the Grand Inquisitor’s enmity.

At midnight Carlo awaits the Queen in her gardens in Madrid, following the instructions in a letter written, he believes, by Elizabeth but in reality penned by Eboli, who mistakenly thinks Carlo loves her. When the veiled Eboli enters, Carlo passionately declares his love; when she unveils, both realize their error. Furiously she accuses him of loving the Queen. Rodrigo comes upon them, grasps the situation and tries to placate Eboli, who runs from the garden swearing to expose Carlo and Elizabeth. To protect the Prince, Rodrigo takes certain incriminating papers from him.

In a square before the Cathedral of Our Lady of Atocha, in Madrid, an immense crowd awaits the appearance of King Philip. The monarch emerges from the church and is greeted by six Flemish deputies led by Carlo. Prince, populace, and court plead for the King’s mercy, but the friars insist on punishment for his rebellious subjects. Drawing his sword, Carlo swears to champion the Flemish cause in defiance of his father, who orders him disarmed. But Carlo surrenders his sword to Rodrigo, while all watch a group of heretics being burned at the stake.

In his study Philip laments his wife’s coldness. He then consults with the Inquisitor, who urges the death of both Carlo and Rodrigo. As the old man leaves, the King muses regretfully that the throne must always yield to the church. Elizabeth bursts in, crying that her jewel casket has been stolen. Philip hands it to her with an ironic demand that she open it. When she hesitates, he breaks the lock, revealing a portrait of Carlo, he accuses her of adultery. The Queen faints as Eboli and Rodrigo enter, the former confessing responsibility for Elizabeth’s betrayal, the latter swearing to free Spain from political oppression. When the men have left, Eboli reveals to the Queen that she gave the casket to Philip from jealousy over Carlo’s love and that she has been the King’s mistress. Dooming Eboli to life in a convent, Elizabeth leaves her. The remorseful princess laments her fatal beauty and swears to save Carlo’s life.

Rodrigo visits Carlo in prison and is shot to death by a minion of the Inquisition. The prince refuses to flee with Eboli but is given his liberty by his father as the furious mob storms into the cell to attack the monarch. Philip is protected by the Grand Inquisitor.

In the monastery cloister, Elizabeth waits to bid farewell to Carlo. The lovers are surprised by the Grand Inquisitor and Philip, who surrenders Carlo into the hands of the Inquisition.