This study explores the effects of chronic administration of vitamin D3 compounds on several biological functions in mice. Knowledge of long-term tolerability of vitamin D3 analogs may be of interest in view of their potential clinical utility in the management of various pathologies such as malignancies, immunological disorders and bone diseases. Four unique vitamin D3 analogs (code names, compounds V, EO, LH and LA) and 1, 25-dihydroxyvitamin D3 (1, 25 (OH) 2D3) were administered ip for 55 weeks to Balb/c mice. Each analog had previously been shown to have potent in vitro activities. After 55 weeks of administration, the mice had a profound decrease in their serum levels of interleukin-2 (IL-2). Likewise, several analogs depressed serum immunoglobulin G concentrations (compounds LH and LA), but levels of blood lymphocytes and splenic lymphocyte subsets (CD4, CD8 and CD19) were not remarkably depressed. The percent of committed myeloid hematopoietic stem cells was 4-to 5-fold elevated in the bone marrow of the mice that received analogs LH and V; nevertheless, their peripheral blood white and red cell counts and platelets were not significantly different in any of the groups. The mice that received 1, 25 (OH) 2D3 had a decrease in bone quantity and quality with a decrease in cross-sectional area and cortical thickness, and a 50% reduction in both stiffness and failure load compared with the control group. In contrast, the cohort that received a fluorinated analog (compound EO) developed bones with significantly larger cross-sectional area and cortical thickness as well as stronger mechanical properties compared with the control group. At the conclusion of the study, body weights were significantly decreased in all experimental mice. Their blood chemistries were normal. Extensive gross and microscopic autopsy analyses of the mice at the conclusion of the study were normal, including those of their kidneys. In conclusion, the vitamin D3 analogs were fairly well tolerated. They did suppress immunity as measured by serum IL-2 and may provide a means to depress the immune response after organ transplantation and for autoimmune diseases. Use of these analogs prevented the detrimental effects of vitamin D3 administration on mechanical and geometric properties of bone, while one analog (compound EO) actually enhanced bone properties. These results suggest that long-term clinical trials with the analogs are feasible.