The microsomal oxidative N-demethylation of N-methylbenzamidine, a model compound for active substances containing the basic amidine function, was investigated. N-Methylbenzamidine was converted into benzamidine and formaldehyde by aerobic incubation with non-induced microsomal fractions of rabbit liver homogenates and NADPH. The formation of benzamidine in the incubation mixtures under widely differing conditions was assayed using a newly-developed, high-performance, ion pair, reverse-phase partition chromatographic method. Optimal reaction conditions were determined. The benzamidine formation in the incubation mixture followed Michaelis-Menten kinetics and required the presence of molecular oxygen and NADPH. The effects of the inducer phenobarbital, methylcholanthrene, ethanol and N-methylbenzamidine itself on the activity were studied. Neither superoxide anion nor hydrogen peroxide was directly involved in the demethylation reaction. The direct involvement of cytochrome P-450 in this reaction is supported by the observation that the presence of inhibitors of cytochrome P-450, in particular of carbon monoxide, markedly decreased the rate of N-demethylation. This N-demethylation of N-methylbenzamidine proves the hypothesis that benzamidines with hydrogen atoms in the alpha-position to the amidine nitrogen atoms are N-dealkylated instead of N-oxygenated by the microsomal mixed function oxidase system.