The oxidative metabolism of losartan (DuP 753), a novel angiotensin II receptor antagonist, by human liver microsomes and purified cytochrome P450 (P450) enzymes, was studied. The primary route of metabolism of losartan is by oxidation of the C5-hydroxymethyl to the carboxylic acid (EXP3174), which is an active metabolite of losartan. When microsomes prepared from different human liver samples were compared, EXP3174 formation activity was well correlated (r2 = 0.93) with nifedipine oxidation (a marker of P4503A4), but not with markers for other human liver P450s. Microsomal oxidation of losartan to EXP3174 was markedly inhibited by gestodene and ketoconazole, selective inhibitors of P4503A enzymes, but not by any of several other P450 inhibitors. Antibodies raised against P4503A4 could inhibit most of the oxidation of losartan to EXP3174 in a microsomal sample having high catalytic activity, but antibodies recognizing other P450s had no effect. The oxidation of losartan to EXP3174 was catalyzed by purified human liver microsomal P4503A4 and by purified bacterial recombinant P4503A4. These results provide evidence that P4503A4 (and possibly other P4503A enzymes) play a major role in the formation of an active metabolite EXP3174.