The small intestinal metabolism of tacrolimus, which is used as an immunosuppressant in transplantation medicine, was investigated in this study. Tacrolimus was metabolized in vitro by isolated human, pig, and rat small intestinal microsomes. The metabolites generated were identified by HPLC/MS. Tacrolimus and its metabolites were quantified using HPLC or HPLC/MS. The cytochrome P450 (CYP) enzymes responsible for tacrolimus metabolism in small intestine were identified using specific CYP antibodies and inhibitors. For characterization of the interindividual variability, microsomes were isolated from small intestinal samples of patients who had undergone resection for various reasons. In an in vitro model using pig small intestinal microsomes, 32 drugs were analyzed for their interactions with tacrolimus metabolism. After incubation with human, rat, and pig small intestinal microsomes, the metabolites 13-O-demethyl and 13,15-O-demethyl tacrolimus were identified. The metabolism of tacrolimus by human small intestine was inhibited by anti-CYP3A, troleandomycin, and erythromycin, indicating that, as in the liver, CYP3A enzymes are the major enzymes for tacrolimus metabolism in the human small intestine. Metabolism of tacrolimus by small intestinal microsomes isolated from 14 different patients varied between 24 and 110 pmol/13-O-demethyl tacrolimus/min/mg microsomal protein, with a mean +/- SD of 54.2 +/- 29.2 pmol/min/mg. Of 32 drugs tested, 15 were found to inhibit small intestinal tacrolimus metabolism: bromocryptine, corticosterone, cyclosporine, dexamethasone, ergotamine, erythromycin, ethinyl estradiol, josamycin, ketoconazole, nifedipine, omeprazole, progesterone, rapamycin, troleandomycin, and verapamil. All of these drugs inhibited tacrolimus metabolism by human liver microsomes as well. It is concluded that tacrolimus is metabolized by cytochrome CYP3A enzymes in the small intestine. The rate of the CYP3A enzymatic activities varies about 5 times from patient to patient, and drugs that interfere with the in vitro metabolism of tacrolimus in the liver also inhibit its small intestinal metabolism.