The interactions of the anticonvulsant drug milacemide (2-n-pentylaminoacetamide) with rat liver mitochondrial monoamine oxidases-A and -B have been studied. The compound acts as a substrate for the B-form of the enzyme, with an apparent Km value of 49 +/- 4.7 microM and a Vmax value of 1.1 +/- 0.2 nmol/min/mg. It is also a time-dependent irreversible inhibitor of that enzyme. Any activity of monoamine oxidase-A towards this substrate was too low to allow accurate determinations to be made by either luminometric determination of H2O2 formation or spectrophotometric coupling of aldehyde formation to NAD+ reduction in the presence of aldehyde dehydrogenase. Milacemide was a reversible competitive inhibitor towards monoamine oxidase-A. The inhibitor constant (Ki) was 115 +/- 35 microM indicating a higher affinity than that towards monoamine oxidase-B, which was also competitively inhibited in the absence of enzyme-inhibitor preincubation (Ki = 331 +/- 185 microM). Determination of the formation of H2O2 and the aldehyde product of the oxidative cleavage of milacemide by purified monoamine oxidase-B from ox liver indicated that cleavage resulted solely in the formation of pentanal and glycinamide. There was no evidence for alternative cleavage to pentylamine and oxamaldehyde.