The rate of formation of the three initial metabolites of cyclosporine metabolism has been determined in liver microsomes of 15 kidney transplant donors. Interindividual variation in metabolite formation was considerable but all three metabolites varied in parallel. An antiserum raised against a steroid-inducible rat cytochrome P-450 (P-450 PCN) strongly inhibited the formation of these metabolites. Immunoquantitation of the protein recognized by a monoclonal antibody reacting with human cytochromes P-450 of the P-450III gene family, homologues of rat P-450 PCN and rabbit P-4503C, revealed a high degree of correlation with microsomal cyclosporine metabolism. The data suggest that this cytochrome P-450 is the major cyclosporine-metabolizing enzyme in human liver. The substrate specificity and the known inducers and inhibitors of this cytochrome P-450 explain several clinically observed drug interactions with cyclosporine.