Highly purified cytochrome P-450 reductase (also called cytochrome C reductase; EC 1.6.2.4.) and NADPH were found to generate superoxide radical (O2.-) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy (ESR) using the spin trapping method with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The signal intensity of DMPO-OOH(-O2.-) (i.e., the DMPO spin adduct of O2.-) was strongest for 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ). O2.- generation with HCAs was decreased in the following order: 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) = 2-amino-3-methylimidazo[4,5-fl-quinoline (IQ) > 2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline (diMeIQx) > or = other HCAs; O2.- generation was lowest with 2-amino-3-methyl-9H-pyrido[2,3-b]indole-CH3COOH (MeA alpha C). By using Lineweaver-Burk plots, Km values of cytochrome P-450 reductase for mitomycin C, IQ, and MeIQ were determined to be 1.60 x 10(-6)M, 1.97 x 10(-5)M, and 2.83 x 10(-6) M, respectively. The N-hydroxyl derivative of 3-amino-1-methyl-5H-pyrido[4,3-b]-indole-HCI-CH3COOH (Trp-P-2), which is considered to be a key intermediate in the reaction of Trp-P-2 with cytochrome P-450 systems, yielded very little generation of O2.-. Because of the known presence of cytochrome P-450 reductase in the cell at significant amount, the present findings are an important implication for carcinogenesis and tumor promotion due to the known effect of oxygen radicals on promotion/progression, cell kill and proliferation.