The capacity of two human hepatic UDP-glucuronosyltransferase (UGT) isoforms, UGT2B7 and UGT2B11, to metabolize more than 50 hydroxylated androgens and pregnanes was investigated. All mono- and dihydroxylated androgens with a hydroxyl function in the 3 alpha, 6 alpha, and 17 beta positions were glucuronidated by UGT2B7, but highest activity was generally observed for steroids containing a 3 alpha-hydroxy substituent. UGT2B7 did not glucuronidate 2 alpha-, 2 beta-, 3 beta-, 6 beta-, 7 alpha-, 11 alpha-, and 11 beta-monohydroxylated androgens, although the presence of hydroxyl groups at certain of these positions did not abolish the ability of UGT2B7 to metabolize diols which also possessed a 3 alpha- or 17 beta-hydroxyl group. 3 alpha-Hydroxypregnanes were also glucuronidated by UGT2B7. Activity was not detected for 6 alpha-, 6 beta-, 11 beta-, 12 alpha-, 16 alpha-, 17 alpha-, 20 alpha-, or 21-monohydroxylated pregnanes. Although 11 alpha-hydroxylated androgens were not glucuronidated by UGT2B7, this enzyme exhibited high activity toward the 11 alpha-hydroxylated derivatives of 5 beta-prenanedione and progesterone. UGT2B11 similarly glucuronidated 3 alpha-hydroxyandrogens and -pregnanes, but rates of metabolism were low compared to UGT2B7. With the exception of androsterone and its 5 beta-isomer, ring A/B stereochemistry appeared not to influence rates of hydroxysteroid glucuronidation by UGT2B7 and UGT2B11. Overall, the data indicate a high degree of stereo- and regioselectivity in the glucuronidation of hydroxyandrogens and -pregnanes by UGT2B7 and UGT2B11 and further suggest that UGT2B7 may contribute to the glucuronidation of 3 alpha-hydroxysteroids in humans.