Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.