Previous work demonstrated that the mitochondrial fraction of rat seminiferous tubules is capable of metabolizing 1,3-dinitrobenzene, using NADPH as a cofactor. Moreover, 1,3-dinitrobenzene treatment of rat tubules caused a decrease in mitochondrial GSH levels. In situ mitochondrial metabolism of 1,3-dinitrobenzene may have caused this depletion through the production of reactive oxygen intermediates, generating oxidative stress and/or one or more metabolites of 1,3-dinitrobenzene which reacted nonenzymatically with GSH. The goal of this study is to investigate which of these two potential mechanisms may have caused the observed GSH depletion. Liver microsomes, known to rapidly metabolize 1,3-dinitrobenzene, generated the superoxide anion radical when incubated with 1,3-dinitrobenzene and NADPH. However, with the seminiferous tubule mitochondria, no oxygen radicals were detected. Hence, the aforementioned GSH depletion is unlikely due to the production of reactive oxygen intermediates from in situ mitochondrial metabolism of 1,3-dinitrobenzene. To investigate the ability of 1,3-dinitrobenzene metabolites to deplete seminiferous tubule mitochondrial GSH, mitochondria were incubated with 1,3-dinitrobenzene and NADPH. Loss of GSH correlated with the appearance of the 1,3-dinitrobenzene metabolites, nitrophenylhydroxylamine and nitroaniline. Subsequent investigation demonstrated that the metabolites, nitrosonitrobenzene, known to react nonenzymatically with nonprotein sulfhydryls, and nitrophenylhydroxylamine both oxidized seminiferous tubule mitochondrial GSH. Further studies suggested that nitrophenylhydroxylamine could deplete GSH via a free radical mechanism. In aqueous solution, this metabolite was shown to exist in equilibrium with a radical form, thought to be the hydronitroxide radical. The addition of GSH eliminated the signal, implying that the radical reacted nonenzymatically with GSH. In conclusion, the data in this study suggest that the decrease in mitochondrial GSH observed in DNB-treated seminiferous tubules is due to the formation of NPHA and NNB and not reactive oxygen intermediates.