4-14C-Estradiol and its 17 beta-sulfate were incubated with rat liver microsomes under NADPH-generating system. Estradiol in liver microsomes from male and female rats was metabolized to multiple kinds of oxidized products including estrone, 2-hydroxyestrone, 2-hydroxyestradiol, and other minor steroids. Incubation of estradiol 17 beta-sulfate was carried out by the same condition, and it was shown that the metabolic pattern between male and female rats was different. By incubation of estradiol 17 beta-sulfate with male rat liver microsomes, 2-hydroxyestradiol 17 beta-sulfate was obtained as the sole product (6%). The hydroxylation was shown to occur without cleavage of the conjugate group. No such regulating effect by conjugate group on 2-hydroxylation of estradiol 17 beta-sulfate was observed in liver microsomes from female rats. The amount of 2-hydroxyestradiol 17 beta-sulfate formed was only 1%, and other metabolites which were thought to be monohydroxylated estradiols were produced as the major products. The 2-hydroxylated metabolite of estradiol 17 beta-sulfate was confirmed by its isolation as a stable form of derivative by the following way. The incubation mixture of massive amount of estradiol 17 beta-sulfate was extracted with n-butanol. Methylation of the extract with diazomethane, followed by acid-catalized hydrolysis, acetylation, and finally separation by preparative thin-layer chromatography, gave a crystalline material, the spectral properties of which were completely identical with those of the synthetic specimen, 2, 3-dimethoxy-1,3,5(10)-estratrien-17 beta-yl acetate.