Highly purified cytochrome P-450 reductase (also called cytochrome c reductase; EC 18.104.22.168.) and NADPH were used to generate superoxide radical (O2) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy using the spin trapping method with 5,5-dimethyl-1-pyrroline JV-oxide (DMPO). The signal intensity of DMP0-OOH(-O2−) (i.e., the DMPO spin adduct of -O2−) was strongest for 2-amino-3,4-dimethy]imidazo[4,5-f]quinoline (MeIQ). The O2− generation with HCAs decreased in the following order: 2-amino-3,8-dimethyI-imidazo[4,5-f] quinoxaline (MeIQx) = 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) > 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (diMeIQx) ≥ other HCAs; O2− generation was lowest with 2-amino-3-methyl-9H-pyrido[2,3-b]indole CH3COOH (MeAαC). By using Lineweaver-Burk plots, Km values of cytochrome P-450 reductase for mitomycin C., IQ, and MeIQ were determined to be 1,60 × 10−6 M, 1.97 × 10−5M, and 2.83 × 10−6 M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.