We systematically characterized the NADPH-dependent metabolites of 17-estradiol and estrone formed by fifteen human cytochrome P450 (CYP) isoforms. CYP1A1 had high activity for estradiol 2-hydroxylation, followed by 15-, 6-, 4-, and 7-hydroxylation. However, when estrone was the as substrate, CYP1A1 formed more 4-hydroxyestrone than 15- or 6-hydroxyestrone, with 2-hydroxyestrone as the major metabolite. CYP1A2 had the highest activity for the 2-hydroxylation of both estradiol and estrone although it also had activity for their 4-hydroxylation (9-13% of 2-hydroxylation). CYP1B1 mainly catalyzed the formation of catechol estrogens, with 4-hydroxyestrogens predominant. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2D6 each showed a varying degree of low catalytic activity for estrogen 2-hydroxylation, whereas CYP2C18 and CYP2E1 did not show any detectable estrogen-hydroxylating activity. CYP3A4 had strong activity for the formation of 2-hydroxyestradiol, followed by 4-hydroxyestradiol and an unknown polar metabolite, and small amounts of 16- and 16-hydroxyestrogens were also formed. The ratio of 4- to 2-hydroxylation of estradiol or estrone with CYP3A4 was 0.22 or 0.51, respectively. CYP3A5 had similar catalytic activity for the formation of 2- and 4-hydroxyestrogens. Notably, CYP3A5 had an unusually high ratio of 4- to 2-hydroxylation of estradiol or estrone (0.53 or 1.26, respectively). CYP3A4 and 3A5 also catalyzed the formation of nonpolar estrogen metabolite peaks (less polar than estrone). CYP3A7 had a distinct catalytic activity for the 16-hydroxylation of estrone, but not estradiol. CYP4A11 had little catalytic activity for the metabolism of estradiol and estrone. In conclusion, many human CYP isoforms are involved in the oxidative metabolism of estradiol and estrone, with a varying degree of catalytic activity and distinct regioselectivity.