(R,S)-Oxazepam is a 1,4-benzodiazepine anxiolytic drug that is metabolized primarily by hepatic glucuronidation. In previous studies, S-oxazepam (but not R-oxazepam) was shown to be polymorphically glucuronidated in humans. The aim of the present study was to identify UDP-glucuronosyltransferase (UGT) isoforms mediating R- and S-oxazepam glucuronidation in human liver, with the long term objective of elucidating the molecular genetic basis for this drug metabolism polymorphism. All available recombinant UGT isoforms were screened for R- and S-oxazepam glucuronidation activities. Enzyme kinetic parameters were then determined in representative human liver microsomes (HLMs) and in UGTs that showed significant activity. Of 12 different UGTs evaluated, only UGT2B15 showed significant S-oxazepam glucuronidation. Furthermore, the apparent K(m) for UGT2B15 (29-35 microM) was similar to values determined for HLMs (43-60 microM). In contrast, R-oxazepam was glucuronidated by UGT1A9 and UGT2B7. Although apparent K(m) values for HLMs (256-303 microM) were most similar to UGT2B7 (333 microM) rather than UGT1A9 (12 microM), intrinsic clearance values for UGT1A9 were 10 times higher than for UGT2B7. A common genetic variation results in aspartate (UGT2B15*1) or tyrosine (UGT2B15*2) at position 85 of the UGT2B15 protein. Microsomes from human embryonic kidney (HEK)-293 cells overexpressing UGT2B15*1 showed 5 times higher S-oxazepam glucuronidation activity than did UGT2B15*2 microsomes. Similar results were obtained for other substrates, including eugenol, naringenin, 4-methylumbelliferone, and androstane-3alpha-diol. In conclusion, S-oxazepam is stereoselectively glucuronidated by UGT2B15, whereas R-oxazepam is glucuronidated by multiple UGT isoforms. Allelic variation associated with the UGT2B15 gene may explain polymorphic S-oxazepam glucuronidation in humans.