The metabolism of chlorpromazine by microsomes in vitro has been examined with extracts from normal liver and small intestinal mucosa of man and guinea pigs. A GC-MS approach has been utilized to measure primary metabolites generated by these extracts, including the S-oxide, N-oxide, 7-hydroxyl, desmethyl, and didesmethyl species. In short term incubations (less than 30 min), the measured metabolites accounted for at least 90% of the substrate utilized. Chlorpromazine metabolism differed strikingly both between species and between hepatic and intestinal tissues of the same species. Guinea pig hepatic microsomes were the most active of the preparations studied, producing relatively large amounts of N-oxide. By contrast, human hepatic microsomes produced the 7-hydroxyl metabolite predominantly, with minimal formation of N-oxide. Extracts of guinea pig intestinal mucosa formed the desmethyl and S-oxide products; an extract of duodenal mucosa from a healthy accident victim exhibited minimal metabolism of chlorpromazine. The kinetics of metabolite formation and studies with inhibitors of cytochrome P-450 suggested the involvement of multiple microsomal enzymes in chlorpromazine metabolism.