Substrate preference of estrogen 2- and 16 alpha-hydroxylases in male rat liver microsomes were determined by using estrone (E1) and estradiol (E2). Kinetic parameters of the hydroxylations showed that E2 is a better substrate for the hydroxylations than E1 and the preferences principally depend on the differences in Vmax values of the hydroxylations between the two substrates. Kinetic studies of inhibition of E2 2- and 16 alpha-hydroxylase activities in the male liver microsomes with ethynylestradiol (EE) and norethisterone (NET), the active ingredients in oral contraceptives, and 17 beta-cyano-16 alpha, 17 alpha-epoxy-1,3,5(10)-estratrien-3-ol were extensively carried out. All of the steroids competitively blocked the two activities and the 16 alpha-hydroxylation was preferentially inhibited by them. Kinetic data, the apparent Ki's for the inhibitors and the apparent Km's for the substrate E2 in the assay, demonstrated that EE and NET are very potent inhibitors for the 16 alpha-hydroxylase. On the other hand, when female rat liver microsomes were used as an enzyme source in the inhibition experiments, the two hydroxylase activities were not so affected by EE and NET and the preferential inhibition was not observed with the ethynyl steroids.