When estradiol 17-sulfate (ES) was incubated with human placental microsomes under an NADPH-generating system, 2- and 4-hydroxyestradiol 17-sulfates (2- and 4-OH-ES) formed as the main products. By kinetic experiments, the apparent Km values of 44.0 and 360 microM and apparent Vmax values of 236 and 140 pmol/mg protein/10 min were obtained for the 2- and 4-hydroxylation, respectively. The results indicate that human placental microsomes have fairly high 2- and 4-hydroxylase activities toward ES. This paper describes the formation of 2- and 4-OH-ES and speculates on their physiological role during pregnancy.