The effects of two anticonvulsant drugs, phenytoin and sodium valproate, on the bioactivation of vitamin D₃ have been studied with respect to the microsomal and mitochondrial cytochrome P-450-linked monooxygenase systems that contribute to 25-hydroxylation of vitamin D₃ in rabbit liver, and the mitochondrial cytochrome P-450-linked monooxygenase system that catalyzes 1α-hydroxylation of 25-hydroxyvitamin D₃ in rabbit kidney. These anticonvulsant drugs were found to inhibit the 25-hydroxylase activity on vitamin D₃ in liver microsomes and mitochondria, respectively, but not to inhibit the 1α-hydroxylation of 25-hydroxyvitamin D₃, even over a wide concentration range. Moreover, the activities of the components of the cytochrome P-450-linked monooxygenase systems: NADPH—cytochrome P-450 reductase, NADP—ferredoxin reductase and ferredoxin, were never inhibited by these drugs. It is possible that the inhibition of bioactivation of vitamin D₃ by these anticonvulsant drugs causes rickets and osteomalacia, and the site of inhibition is expected to be the cytochrome P-450 mediated reactions in liver mitochondria.