BACKGROUND Cardioplegic arrest and reperfusion of the myocardium imposes oxidative stress that could potentially inactivate metabolic enzymes and compromise energy production. This study determined the impact of cardioplegic arrest and reperfusion on activities of several oxidant-sensitive enzymes, and tested whether pyruvate, a natural metabolic fuel and antioxidant, mitigates oxidant stress, protects enzymes, and bolsters myocardial energy state after reperfusion. METHODS In situ swine hearts were arrested for 60 minutes with 4:1 blood:crystalloid cardioplegia, and then reperfused for 3 minutes with cardioplegia-free blood with or without approximately 12 mM pyruvate. Tissue metabolites and enzyme activities were measured in left ventricular myocardium snap frozen at 45 minutes of arrest and 3 minutes of reperfusion. RESULTS The 8-isoprostane content, a measure of lipid peroxidation, sharply increased upon reperfusion, coincident with a 70% decline in redox state of the intracellular antioxidant glutathione. Aconitase and glucose 6-phosphate dehydrogenase activities fell during arrest; creatine kinase and phosphofructokinase were inactivated upon reperfusion. Pyruvate suppressed 8-isoprostane formation, maintained glutathione redox state, and enhanced phosphocreatine phosphorylation potential, a measure of myocardial energy state, during reperfusion. Pyruvate reactivated creatine kinase and aconitase, which are at least partially mitochondrial enzymes, but did not protect the cytosolic enzymes glucose 6-phosphate dehydrogenase and phosphofructokinase. CONCLUSIONS Administration of pyruvate upon reperfusion after cardioplegic arrest mitigates oxidative stress, protects mitochondrial enzymes and increases myocardial energy state. These results support therapeutic application of pyruvate-enhanced reperfusion to prevent cardiac injury after cardioplegic arrest.