The number of two-toed sloths (Choloepus hoffmanni) has significantly decreased in the last years. Deepening the knowledge of this tropical mammal's reproductive physiology is essential to improve captive breeding within conservation programs for this species. However, several aspects of its reproductive biology remain unexplored and have not been described sufficiently. The aim of this work was to describe the estrous cycle and reproductive physiology of two adult female C. hoffmanni by vaginal cytology, appearance of the external genitalia, and behavior. Vaginal cytology assay showed that the average duration of the estrous cycle was 15.1 ± 4.53 d. Positive correlations (P < 0.05) were found between the peak presence of superficial cells (estrous phase) and four parameters: aggressive behavior, pursuing behavior, vulvar swelling, and vaginal discharge. This pilot study, conducted on just two animals, forms a basis for a study design that may be employed for a more comprehensive assessment of the two-toed sloth reproductive physiology and behavior.