The contribution of the adrenals, the testes, and a light/dark cycle to the regulation of hepatic steroid hydroxylases was studied in vitro by measuring transformation rates (nmol/mg protein/min) of dehydroepiandrosterone (DHA). Normal and adrenalectomized male rats were kept in 12 h light:12 h dark (12L:12D) or in constant light (CL) for 1 week. Normal and castrated rats were kept in 12L:12D for 3 weeks. Rats were killed at 4-h intervals over a 36-h period, hepatic microsomal fractions were incubated with [4-14C]DHA, and C-7 and C-16-oxygenated products were quantitated. The overall mean rate of 7alpha-hydroxylation decreased in normal rats exposed to CL. Rates of both 7alpha- and 16alpha-hydroxylation were significantly greater in normal rats than in the respective group of treated rats, except for the 7alpha-hydroxylase in castrated rats. These results indicate that 16alpha-hydroxylase activity is both gonadal- and adrenal-dependent, whereas 7alpha-hydroxylase activity is adrenal-dependent only. Both enzymes are sensitive to light/dark modulations, although a circadian rhythm could not be conclusively established.