According to biographer Barry Paris, the monkey was supposed to be Sister Luke's beloved pet, but gave Audrey a nasty bite on the arm.

The role of Sister Luke in The Nun's Story would be Audrey's greatest challenge as an actress and make a lasting impression on her soul, planting the seed of a calling that would take her back to Africa thirty years later as a Goodwill Ambassador to UNICEF. The film was based on the novel of the same name by Kathryn Hulme, which told the true story of Belgian nun Marie-Louise Habets, who served as a nurse in the Belgian Congo. Her internal struggles with her vow of obedience, particularly where it concerned her religious duty to aid Nazi soldiers despite the murder of her father, eventually led her to leave the order. Audrey's old friend Gary Cooper had sent a copy of the novel to director Fred Zinnemann, who persuaded Warner Bros. to take on the challenging project with the promise of Hepburn in the lead role.

Audrey became very close to Habets and Hulme, consulting the real life Sister Luke on every aspect of her character, keen to represent accurately the details of Catholic rituals and convent life. Her intense attention to detail is evidenced in the various lists, schedules and other documents that she studied and annotated in preparation for the role . Zinnemann arranged for Audrey and other key actresses to oversee medical procedures and even to live in a convent alongside real nuns for several days, recalling I stashed my 'nuns' away at different convents... all of them would come out of the cloisters absolutely purple with cold but fascinated by what they were involved in and very excited by the way they were getting prepared for their characters. The biggest challenge in getting the film made was securing the cooperation of the Catholic church and avoiding censure of the film for its treatment of the controversial subject matter. He later recalled All film companies approaching the Catholic Church for assistance are assigned someone ... to work with them... In our case they were extremely thorough in scrutinising our shooting script. Hours were spent on negotiations over a single word, though gradually the Dominicans came to trust us and generously assist with the production.

Shooting began on location in the Belgian Congo in January through to March 1958, when the company returned to Rome to shoot the interior scenes. Audrey found a lot of similarities between herself and Sister Luke - both had been born in Belgium, had been caught up in the war in Holland, had family members captured by the Germans and both had lost their fathers. She became so intensely involved in the character that it became her reality, she was no longer just playing at being a nun, telling the press the part was suited to my nature, and later explaining I found that something happened when I put on the habit of a nun. Once you do that, you feel something. Writing to Hulme and Habets from Rome, Audrey described the deep impressions made on her by the nuns she met in Paris and Africa and how the experiences had altered her forever I am and feel a different person. Audrey's companion Robert Wolders later told biographer Barry Paris I think Audrey was much more comfortable with Sister Luke than with other parts... It was the story of a woman who investigated life, who was constantly on a search, as Audrey was.

Towards the end of production, Zinnemann wrote to Hepburn's agent Kurt Frings I have never seen anyone more disciplined, more gracious or more dedicated to their work than Audrey... she has proven herself a great actress in a very difficult and exacting part. Critics praised the depth and complexity of her performance, with Films in Review magazine declaring it one of the great performances of the screen. The film was nominated for eight Oscars, including Best Actress for Hepburn, but won none, the MGM epic Ben Hur sweeping the board. Nevertheless, The Nun's Story was the most financially successful Warner Bros. production to date and Hepburn was recognised as Best Actress of 1959 by the New York Film Critics and the British Academy.
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