We studied the action of the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene (CDNB) on the synaptosomal production of H2O2. We found that CDNB (30-40 microM) readily depletes the cytosolic glutathione but is almost without effect on the mitochondrial fraction. The depletion of the cytosolic glutathione induced by CDNB affords the detection in the extracellular space of H2O2 produced intrasynaptosomally upon increasing the cytosolic Ca2+ concentration that is otherwise destroyed by glutathione peroxidase. Higher concentrations of CDNB induce a H2O2 production which is not related to the glutathione content. This H2O2 is of mitochondrial origin and requires that NAD be reduced. The primary product of the mitochondrial CD-NB-dependent oxygen reduction is at least in part the superoxide anion.