Two-toed sloths (Choloepus hoffmani, C. didactylus) are unique animals popular in zoological settings. The role of vitamin D in the maintenance of calcium homeostasis in this species is largely unexplored, although disorders of calcium homeostasis have been reported in managed sloths, including urolithiasis and soft tissue mineralization. We have performed some preliminary investigations on this topic in C. hoffmanni, evaluating a new laboratory method for serum vitamin D measurement, investigating the capacity for cutaneous photobiosynthesis of vitamin D, and a pilot study correlating vitamin D and mineral values in serum and diet in a zoo-housed group of 9 sloths. The research project outlined in this proposal will further characterize the vitamin D status of C. hoffmanni in the United States (U.S.) by looking at serum values in correlation with diet across multiple zoological institutions, assessing differences in husbandry practices on these parameters. Our goal is to utilize this information to better inform husbandry and nutritional practices for C. hoffmanni, and reduce morbidity and mortality related to disorders of calcium homeostasis in this species.