The neuroleptic agent haloperidol (HP) and its tetrahydropyridine dehydration product HPTP are biotransformed to the potentially neurotoxic HP pyridinium species HPP+ and the reduced HP pyridinium species RHPP+ in humans and rodents. The studies reported here were designed to identify the specific form(s) of human cytochrome P450 that catalyze(s) these transformations. Fifteen human liver microsomal preparations all catalyzed the oxidation of HP and HPTP to HPP+ and HPTP to RHPP+. Values for kcat/KM averaged 6.71 and 1.24 min-1 mM-1 for HPP+ and RHPP+ formation, respectively. The rates of conversion of HP and HPTP to HPP+ correlated well with testosterone 6 beta-hydroxylase activity, a marker of P450 3A activity. Microsomes prepared from a human lymphoblastoid cell line co-expressing human P450 3A4 and cytochrome P450 reductase also catalyzed the formation of HPP+ from HP and HPTP. Troleandomycin and ketoconazole, potent P450 3A inhibitors, and antibodies against P450 3A were effective inhibitors of HPP+ formation. We conclude that the conversions of HP and HPTP to potentially neurotoxic pyridinium metabolite HPP+ are catalyzed selectively by P450 3A4 in human liver microsomes.