A concentration-dependent increase in the generation of the superoxide anion radical (O-2), was observed during the incubation of benzidine (0.1-5 mM), but not of the structurally related compounds 4-aminobiphenyl, 2-aminobiphenyl or 4-fluoro-4'-aminobiphenyl, with NADPH-supplemented rat liver microsomes. This increase was partially inhibited by carbon monoxide and catalase but unaffected by 4-aminobiphenyl, a substrate of the cytochrome P-450 system. Microsomes from benzo(a)pyrene-treated, but not microsomes from phenobarbitone-pretreated rats, were responsible for a larger benzidine-dependent effect compared to microsomes from control animals. In contrast to the above observations, benzidine decreased the formation of hydrogen peroxide by NADPH-supplemented microsomal preparations from untreated rats. These results indicate that free radicals of oxygen are generated during the metabolism of some arylamines.