The metabolism of stiripentol (I), a new antiepileptic drug, was studied in healthy human subjects. Following a single 1200-mg oral dose to one subject, 13 metabolites of I were detected in urine and were identified by GC/MS techniques. The structures of 9 of these metabolites were confirmed subsequently by synthesis of the corresponding reference compounds. The nature of the urinary metabolites of I revealed the operation of five distinct metabolic pathways for this drug, viz. conjugation with glucuronic acid, oxidative cleavage of the methylenedioxy ring system, O-methylation of catechol metabolites, hydroxylation of the t-butyl group, and conversion of the allylic alcohol side-chain to the isomeric 3-pentanone structure. Metabolites of I excreted into urine over 12 hr accounted for the majority (73%) of an acute dose, whereas a further 18% was recovered in feces as the unchanged drug. These findings suggested that the search for additional metabolites would yield only trace amounts. From a quantitative standpoint, the most important pathway of biotransformation of I following both acute and chronic dosing involved opening of the methylenedioxy ring to generate catechol derivatives. This finding probably accounts for the known inhibitory effects of I on the oxidative metabolism of other antiepileptic agents and for the clinically significant drug interactions involving stiripentol.