The in vitro metabolism of nefiracetam (CAS 77191-36-7, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, NEF, DM-9384), a novel cognition enhancer, has been investigated using liver microsomes from rats, dogs and monkeys. Microsomal metabolism of NEF showed qualitatively a similar profile in three species tested. Six metabolites were generated from incubation of NEF with liver microsomes. Their structures were identified using a thermospray LC/MS/MS (liquid chromatography/tandem mass spectrometry) method, as regioisomers of monhydroxylated derivatives of NEF: the 3-hydroxy (3-OH-NEF); 4-hydroxy (4-OH-NEF); 5-hydroxy (5-OH-NEF); 3'-hydroxy (3'-OH-NEF); 4'-hydroxy (4'-OH-NEF); hydroxymethyl (HM-NEF) metabolites. The heat lability, NADPH requirement and inhibition by prototype cytochrome P450 inhibitors (proadifen and metyrapone) implies that NEF oxidations are catalyzed by cytochromes P450. The major metabolic route was 5-OH-NEF formation in all species, corresponding well with the previous investigations in vivo. Inhibitory effects of alpha-naphthoflavone and quinidine on NEF hydroxylation suggest that several isoforms of cytochromes P450 are involved in the formation of these NEF metabolites.