The metabolism of radiolabelled alosetron was studied in rat, dog, rabbit, mouse and human. The metabolism in rat and dog was studied at a low and an elevated dose designed to generate sufficient quantities of metabolite for definitive identification. A strategy for the characterization of metabolites in cases of extensive metabolism was developed and demonstrated for alosetron. Semi-preparative high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and liquid chromatography-nuclear magnetic resonance (HPLC-NMR) enabled the isolation and characterization of 28 metabolites of alosetron. The characterization of the metabolites in animal excreta facilitated the identification of human systemic metabolites.