The changes in mitochondrial and microsomal enzyme activities were examined during the course of 2-acetylaminofluorene (2-AAF) hepatocarcinogenesis. 2-AAF produced the phasic changes, such as the early reduction, back to the control and the re-reduction, in mitochondrial monoamine oxidase (MAO) B and kynurenine 3-hydroxylase activities during the course of its hepatocarcinogenic activity in male Wistar rats. 2-AAF also produced decreases in microsomal cytochrome P-450 content and aminopyrine demethylase activity, while it caused an early significant increase and subsequent decrease in cytochrome b5 content in the liver. The change in microsomal protoheme content totally paralleled that of the hemeproteins. Microsomal heme oxygenase activity was significantly increased during 2-AAF feeding, while mitochondrial delta-aminolevulinic acid synthetase activity tended to decrease under the experimental conditions. These changes in mitochondrial and microsomal enzyme activities were more pronounced in the nodules and hepatomas as compared to those of the surrounding tissues.