Cimetidine interaction with cytochrome P-450 may reduce binding affinity of some drugs, causing reduced metabolism of substrates such as diazepam and warfarin. Isoniazid also inhibits hepatic mixed function oxidase activity and has been reported to depress circulatory levels of hydroxylated vitamin D metabolites. Because hepatic vitamin D 25-hydroxylase is considered to be a cytochrome P-450-dependent enzyme, we examined the effect of cimetidine and isoniazid on hepatic vitamin D 25-hydroxylase activity in vitro in the rat. The assay system employed whole liver homogenates from rats deficient in vitamin D and chromatographic separation of radiolabeled substrate and product. Cimetidine and isoniazid were incubated with liver homogenates prior to the addition of 12 nmol/L final concentration of tritiated vitamin D. In addition, assays were performed in rats deficient in vitamin D given cimetidine or isoniazid 1 hour before sacrifice. Both cimetidine and isoniazid inhibited vitamin D 25-hydroxylase activity in vitro. Vitamin D 25-hydroxylase activity was depressed to 75%, 65%, and 55% of control values by cimetidine at 3, 6, and 10 mmol/L concentrations, respectively, and to 81%, 53%, and 35% by isoniazid at 1, 5, and 10 mmol/L. In vivo intraperitoneal administration of 120 mg/kg cimetidine depressed vitamin D 25-hydroxylase activity by 22%, and the same dose of isoniazid inhibited activity by 26%. The effect of long-term cimetidine therapy on vitamin D metabolism requires further evaluation.