3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO(x)·) and superoxide radical (O2/-·). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (<1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O2/· dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metalcatalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.