The enzymatic activity of the mixed-function oxidase system in the kidney and liver was evaluated by means of an in vitro N-demethylation activity assay with aminopyrine as the substrate. Renal and hepatic demethylation activity of 9000 x g supernatant fraction was determined in the rat, rabbit, and guinea-pig. In terms of interspecies comparison, the renal tissue demethylation activities were on a similar level with a slight increase in the order guinea-pig, rabbit and rat. In relation to hepatic activity, these relative demethylation activities of renal tissue had the same values in the rat and rabbit, whereas that in the guinea pig was significantly lower. The distribution of demethylation activity in the kidney was determined by comparing the cortex and medullary activity in relation to the total kidney tissue activity in the rabbit and guinea-pig. Although the higher demethylation activities were obtained in rabbit renal preparations and low demethylation activity was detected in the guinea-pig renal medulla only, no significant interspecies differences were found by the statistical evaluation. It may be concluded that the mixed-function oxidase system responsible for renal demethylation activity seems to be concentrated in the renal cortex and its distribution coincides in the rabbit and guinea-pig kidney.