Alterations in circulating vitamin D3 metabo-lites have been documented in both experimental and human diabetes mellitus. Using a recirculating hepatic perfusion system and in vitro kidney mitochondrial assays, we studied vitamin D3 hydroxylation in control and insulin-deficient rats 6 weeks after the induction of streptozotocin-diabetes. Vitamin D3-25-hydroxylase activity, assessed by hepatic conversion of [3H]vitamin D3 to [3H]25-hydroxyvitamin D3 during a 4-h perfusion, was similar in diabetic and control animals. The hepatic degradation of 25-hydroxyvitamin D3 to more polar metabolites was also normal, as was glucuronide conjugation and biliary excretion of vitamin D3 metabolites. The chronic insulin-deficient state resulted in a significantly (P < 0.01) decreased 1α-hydroxylase activity and enhanced (P < 0.001) renal 24-hydroxylase activity. These alterations in vitamin D metabolism may relate to the deranged mineral homeostasis and skeletal morphology observed in rats and humans with chronic insulin deficiency.