The ability of soybean lipoxygenase to mediate NAD(P)H oxidation and concomitant superoxide generation in the presence of linoleic acid was examined. At optimum pH of 8.3, lipoxygenase oxidized both NADH and NADPH in the presence of 700 microM linoleic acid. The oxidation of NAD(P)H was biphasic. The initial rates of NADH and NADPH oxidation were 130 and 140 nmoles/min/nmole of enzyme respectively and the corresponding final rates were 344 and 350 nmoles/min/nmole of enzyme. The apparent Km values calculated for NADH and NADPH oxidation were 13 microM and 117 microM respectively. NAD(P)H oxidation was accompanied by the reduction of either ferricytochrome c or nitroblue tetrazolium (NBT) which can be abolished (approx. 85%) by superoxide dismutase (SOD) suggesting the generation of superoxide anion radicals. Under optimal conditions, the rates of superoxide generation, measured as the SOD-inhibitable reduction of ferricytochrome c, were 325 and 235 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Under identical experimental conditions, the SOD-inhibitable NBT reduction rates were 308 and 210 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Both NADH and NADPH could be regenerated after oxidation using the appropriate dehydrogenases. These results strongly suggest that lipoxygenase not only generates lipid hydroperoxides but can also generate superoxide via oxidation of pyridine nucleotides and may, therefore, significantly contribute to oxidative stress in cells.