5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.