The opera opens with scenes showing typical aspects of life in the convent – all the sisters sing hymns, the Monitor scolds two Lay-Sisters, everyone gathers for recreation in the courtyard. The sisters rejoice because, as the Mistress of Novices explains, this is the first of three evenings that occur each year when the setting sun strikes the fountain so as to turn its water golden. This event causes the sisters to remember Bianca Rosa, a beloved sister who passed away a year ago to date. Sister Genevieve suggests they pour some of the "golden" water onto her tomb.

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The nuns discuss their desires. While the Monitor believes that any desire at all is wrong, Sister Genevieve confesses that she wishes to see lambs again because she used to be a shepherdess when she was a girl, and Sister Dolcina wishes for something good to eat. Sister Angelica claims to have no desires, but as soon as she says so, the nuns begin gossiping – Sister Angelica has lied, because her true desire is to hear from her wealthy, noble family, whom she has not heard from in seven years. Rumors are that she was sent to the convent as punishment for having a child out of wedlock. 

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The conversation is interrupted by the Infirmary Sister, who begs Angelica to make one of her special herbal remedies to treat a painful wasp sting. Two other sisters arrive, bringing supplies to the convent, and news that a grand coach is waiting outside. Sister Angelica becomes nervous and upset, thinking rightly that someone in her family has come to visit her. The Abbess chastises Sister Angelica for her inappropriate excitement and announces the visitor, the Princess, Sister Angelica's aunt.

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The Princess explains that Angelica's little sister is to be married and that Angelica must sign a document concerning the partition of the family fortune in preparation. Angelica reaffirms her will to expiate her sins, but asks for something in return before signing the document. She asks her aunt to tell her about her illegitimate son, who was taken from her seven years ago at birth and who she hasn't heard anything about since. The Princess at first refuses to speak, but finally informs Sister Angelica that her son died of fever two years ago. Sister Angelica, devastated, signs the document and collapses in tears. The Princess leaves. 

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Sister Angelica is seized by a heavenly vision – she believes she hears her son calling for her to meet him in paradise. Using her knowledge of herbs, she makes a poison and drinks it. To her horror, she realizes that in taking her own life, she has committed a mortal sin and has condemned herself to eternal separation from her son. She begs the Virgin Mary for mercy and, as she dies, she sees a miracle: the Virgin Mary appears to personally carry her soul to heaven, along with Sister Angelica's son, who runs to embrace her.