The metabolism of an oral dose (20 mg) of the antianxiety drug buspirone labeled with 14C/15N was studied in human subjects. 15N was incorporated in the molecule to facilitate structural characterization of the metabolites by mass spectrometry. Urine samples were collected at intervals up to 24 hr and analyzed for radioactivity. Cumulative urinary excretion accounted for 50% of the dose in 24 hr. The urine was hydrolyzed with beta-glucuronidase/arylsulfatase and the deconjugated metabolites were isolated and purified by HPLC. The purified metabolites were identified by GC/MS, 1H-NMR, and comparison with authentic standards when available. Seven metabolites of buspirone were identified unambiguously, together with unchanged drug. Hydroxylation alpha to the glutarimidyl carbonyl at the 6'-position on the spiro ring system, hydroxylation at the 5-position on the pyrimidine ring, and N-dealkylation of the butyl-substituted side chain were major routes of metabolism. The identified metabolites accounted for 88% of the total radioactivity in the urine. A scheme for metabolism of buspirone in human subjects has been proposed.