Anaerobic incubations containing nitrofurantoin, and NADPH-generating system, and chick hepatic microsomes produced an electron spin resonance spectrum identified as the nitro anion free radical. Aerobically, nitrofurantoin markedly stimulated oxygen consumption, superoxide formation, and NADPH oxidation in hepatic microsomal preparations from control and selenium-deficient chicks. The nitrofurantoin-stimulated oxidation of NADPH was inhibited by superoxide dismutase (SOD). The superoxide-dependent oxidation of NADPH did not appear to be mediated by an NADP• radical, as has been shown for lactate dehydrogenase. Further, the aerobic metabolism of the nitro drug was also affected by SOD, suggesting the existence of a previously unreported metabolic pathway for nitrofurantoin. These studies support the growing body of evidence which suggests that nitrofurantoin toxicity is mediated, at least in part, by the metabolic activation of oxygen by the nitro aromatic anion radical. Further, these data suggest that superoxide may be involved in the oxidative metabolism of the aromatic nitro compounds.