9-Nitro-20(S)-camptothecin is a novel anticancer drug. In this study, metabolites of 9-nitro-20(S)-camptothecin in rats were identified. Rats were dosed with the drug, and the metabolites in the bile were isolated and collected by high-performance liquid chromatography using a gradient elution. By LC/MSn(n =1-3), the biliary metabolites in addition to the unchanged drug were identified as 9-amino-20(S)-camptothecin (M3), 9-acetamido-20(S)-camptothecin (M4), and the glucuronide of 9-hydroxy-20(S)-camptothecin (M1) with the aid of the reference substances and the enzymatic hydrolysis. The accurate mass data for metabolites M2 and M5 were determined by a quadrupole time-of-flight mass spectrometer. Metabolite M5 was suggested to be the glutathione conjugate of 10-hydroxy-20(S)-campto-thecin, and M2 may arise from the loss of glutamic acid from M5. Metabolites M1 and M3 were also found in the urine, and M4 in the feces. Fecal metabolite M7 was confirmed to be 9-hydroxy-20(S)-camptothecin (the aglycon of M1) by being compared with the reference standard. The mercapturic acid conjugate of 10-hydroxy-20(S)-camptothecin (M6) was detected in the urine and feces by LC/MSn