The participation of rat hepatic P-450 in the conversion of 17 beta-estradiol to catechol estrogens was examined by means of enzyme reconstitution and immunoinhibition studies. It was thus demonstrated that three rat liver microsomal cytochrome P-450 forms, designated P-450UT-A, P-450PCN-E, and P-450ISF-G, each contribute to the 2- and 4-hydroxylation of 17 beta-estradiol catalyzed by hepatic microsomal preparations. Two of these enzymes, P-450UT-A and P-450PCN-E, are expressed constitutively, are male-specific, and are regulated by testosterone as well as influenced by the administration of various chemicals. Consistent with these observations, 17 beta-estradiol 2- and 4-hydroxylation activities both increased rapidly during puberty in male rats and were induced by treatment of rats with phenobarbital or pregnenolone 16 alpha-carbonitrile. Castration of male rats at birth or at 5 weeks of age suppressed the levels of 17 beta-estradiol 2- and 4-hydroxylase activities measured at 10 weeks of age. This suppression of activity was reversed upon administration of testosterone during the neonatal period (days 1 and 3 of life) or by capsule implantation at 5 weeks of age. These patterns of 17 beta-estradiol 2- and 4-hydroxylation are discussed in terms of the previously characterized response of the multiple rat hepatic P-450 forms to ontogenic, hormonal, and xenobiotic factors.