The activities of estrogen-2,4- and 16α-hydroxylase as well as 17β-hydroxysteroid dehydrogenase (17β-OHSDHase) were determined in normal, benign and mammary tumor breast tissues. All three hydroxylases were absent in both normal and benign tumor breast tissues. 17β-OHSDHase was absent from normal breast tissues but present in all benign tumor tissues. Estrogen-2-hydroxylase was present in most breast carcinomas, was significantly higher in estrogen receptor (ER)-positive than ER-negative tumors, did not correlate with progesterone receptor (PR) and was significantly lower in the ER+PR- subgroup of breast cancers. 4-Hydroxylase activity did not correlate with either ER or PR content but was significantly lower in the ER+PR- subgroup of breast cancers. The activity of 16α-hydroxylase was present in only 18% of all tumors investigated ( 9 50), did not correlate with the ER or PR content and was completely absent in the ER+PR- and ER-PR+ subgroups. The activity of 17β-OHSDHase was significantly higher than the estrogen hydroxylase but did not correlate with either the ER or PR content, and was not different among the ERPR tumor subgroups. The physiological role of these enzymes in the metabolism of estradiol in relation to breast cancer is discussed.