Clonazepam, its nitro-reduced metabolites, and the 3-hydroxyl-ated derivatives, were separated by reverse-phase hplc using a four solvent optimization procudure. The separation was used to quantitate the metabolism of3H-clonazepam in rat liver microsomes under aerobic or anaerobic conditions by collection of HPLC eluents and liquid scintillation counting. The predominant metabolite in aerobic incubations was 3-hydroxyclonazepam. The primary anaerobic metabolite was 7-aminoclonazepam. The identity of the microsomal metabolites was confirmed by chemical ionization mass spectrometry. This separation could be applicable for future metabolic studies of clonazepam.