Superoxide dismutase (SOD) has been championed as an effective antioxidant for the treatment of ischemia-reperfusion injury in a wide variety of tissues. Unfortunately a bell-shaped dose-response curve has been observed, whereby SOD at higher concentrations loses its effectiveness and may even enhance the extent of reperfusion injury. Using the xanthine/xanthine oxidase reaction to generate superoxide radicals, we have attempted to examine the role of the Fenton reaction in SOD toxicity observing that high SOD levels along with micromolar concentrations of Fe2+ greatly increased the production of the highly toxic hydroxyl radical. The production of superoxide radicals and their conversion to hydroxyl radicals were measured by using the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and electron spin resonance (ESR). Attempts to counter the toxicity of SOD involved the covalent conjugation of SOD to catalase in an effort to lower the available free H2O2 and thus minimize the extent of the Fenton reaction. The conjugate was tested in both the xanthine/xanthine oxidase system and a rat heart model of ischemia-reperfusion. In the xanthine/xanthine oxidase model, the combination of SOD and Fe2+ results in an enhanced production of hydroxyl radicals which is inhibited by the inclusion of catalase. In reperfused ischemic hearts, working at levels of free SOD which were either toxic or failed to give any protection against reperfusion injury, an equivalent amount of SOD conjugated to catalase resulted in an 80% return to normal mechanical function of the reperfused hearts. We attribute the toxicity of free SOD in hearts subjected to ischemia-reperfusion injury to the production of hydroxyl radicals as a result of the increased Fenton reaction. This reaction is inhibited by the presence of catalase conjugated to SOD.