1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.