Fluoxetine and its primary metabolite, norfluoxetine, are inhibitors of neuronal uptake of 5-hydroxytryptamine. While fluoxetine has also been reported to inhibit monoamine oxidase (MAO) in vitro at concentrations much lower than those measured in brain following chronic fluoxetine treatment, neurochemical profiles are not consistent with substantial MAO inhibition in vivo. In an attempt to explain this inconsistency, we have examined the interactions of fluoxetine and norfluoxetine with rat brain MAO-A and -B by a radiochemical assay method. Fluoxetine and norfluoxetine were competitive inhibitors of MAO-A in vitro, with Ki values of 76.3 microM and 90.5 microM, respectively. Both compounds were non-competitive or uncompetitive inhibitors of MAO-B in vitro. Inhibition of MAO-B was time-dependent and was very slowly reversible by dialysis. IC50 values versus metabolism of 50 microM beta-phenylethylamine were 17.8 microM (fluoxetine) and 18.5 microM (norfluoxetine). Analysis of the time-dependence of MAO-B inhibition by fluoxetine revealed that an initial competitive interaction between the enzyme and the inhibitor (Ki 245 microM) was followed by tight-binding enzyme inactivation (K(inact) 0.071 min-1). Following administration of fluoxetine (20 mg kg-1 day-1) for 7 days, the cortical concentration of fluoxetine + norfluoxetine was estimated by gas-liquid chromatography to be 700 microM. Such drug treatment reduced MAO-A activity by 23% in 1:8 (w/v) cortical homogenates, but not in 1:80 homogenates. Inhibition of MAO-B in 1:8 homogenates was modest (12%) and was not significantly reduced by homogenate dilution. The concentration of 5-hydroxyindole-3-acetic acid, measured by high pressure liquid chromatography, was reduced by 47% in cortices from drug-treated rats, while concentrations of 5-hydroxytryptamine, noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were unchanged. These results suggest that, following chronic drug administration leading to relatively high tissue concentrations of fluoxetine and norfluoxetine, inhibition of either form of MAO would be restricted by competition for the enzyme with intraneuronal amine substrates.