Disorders of calcium metabolism in sloths occur with regularity, including published reports of soft tissue mineralization possibly related to vitamin D toxicity. Two-toed sloths are unique animals popular in zoo settings, yet both the captive and wild populations are in decline. No research has been published on calcium and vitamin D metabolism in sloths. This proposed research is designed to generate thorough baseline data designed to provide important information regarding vitamin D and calcium metabolism in Hoffmann’s two-toed sloths. The information gained from this proposed research will also provide a foundation for future research projects in this species designed to improve the knowledge about metabolism and physiology in two-toed sloths, reduce the incidence of disease conditions related to calcium homeostasis, and improve their health and welfare in a captive setting.