Three lines of investigation indicated that hydrogen peroxide (H2O2) from xanthine oxidase (XO) contributes to cardiac dysfunction during reperfusion after ischemia. First, addition of dimethylthiourea (DMTU), a highly permeant O2 metabolite scavenger (but not urea) simultaneously with reperfusion improved recovery of ventricular function as assessed by ventricular developed pressure (DP), contractility (+dP/dt), and relaxation rate (-dP/dt) in isolated Krebs-Henseleit-perfused rat hearts subjected to global normothermic ischemia. Second, hearts from rats fed tungsten or treated with allopurinol had negligible XO activities (less than 0.5 mU/g wet myocardium compared with greater than 6.0 mU/g in control hearts) and increased ventricular function after ischemia and reperfusion. Third, myocardial H2O2-dependent inactivation of catalase occurred after reperfusion following ischemia, but not after ischemia without reperfusion or perfusion without ischemia. In contrast, myocardial catalase did not decrease during reperfusion of ischemic hearts treated with DMTU, tungsten, or allopurinol.