A female patient was treated with irinotecan (CPT-11) for liver metastatic colon carcinoma. She had a percutaneous biliary catheter because of extrahepatic biliary obstruction. The patient was treated with CPT-11 for three courses at doses of 350 mg/m2 for the first course and 300 mg/m2 for the remaining courses, given as a 30-min i. v. infusion. Metabolism studies in bile and urine were performed by coupling high-performance liquid chromatography to electrospray mass spectrometry. Conventional spectra [liquid chromatography/mass spectrometry (LC/MS)] allowed on-line molecular mass determination of CPT-11 and its main metabolites, whereas structural information was obtained by tandem mass spectrometry (LC/MS/MS). At least 16 metabolites were detected in bile, while 8 of them were also detected in urine. Three compounds were identified as the parent drug, the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), and SN-38 glucuronide. The major metabolic pathway consists in oxidations of the terminal piperidine ring of the CPT-11 side chain, which eventually results in the formation of a primary amine. Other metabolites result from oxidation of the camptothecin nucleus. Finally, decarboxylation of the carboxylate form of CPT-11 was also observed. Several metabolites result from combinations of these pathways. The structures of the identified metabolites indicate for the first time a major role of monooxygenases in the elimination of a camptothecin derivative in humans. This finding will allow better understanding of interindividual variability in pharmacokinetics and intestinal toxicity of CPT-11.