This study assesses the ability of the free-radical scavenger peroxidase to enhance cardioplegic protection when given during or before myocardial ischemia. Forty-four isolated isovolumetric buffer-perfused rat hearts were studied. In a first series of experiments that consisted of three groups, hearts were subjected to 90 min of normothermic global ischemia followed by 45 min of reperfusion. One group received a crystalloid cardioplegic solution given as a single dose at the onset of arrest. A second group received cardioplegic solution supplement with superoxide dismutase (200,000 U/liter), and a third group received cardioplegic solution supplement with peroxidase (6000 U/liter). Based on comparisons of postreperfusion coronary flow, left ventricular developed pressure, maximum dP/dt, and diastolic pressure, we found that the best protection was provided by peroxidase-enriched cardioplegia. A second series of experiments was then undertaken to asses the effects of the latter enzyme given as a pretreatment. Hearts were subjected to 3 hr of global ischemia, during which myocardial protection was provided by hypothermia (15° C) along with multidose cardioplegia. The treatment group was given peroxidase (10,000 U/liter) added to the perfusate fluid for 15 min before the onset of cardioplegic arrest without further enzyme supplementation during ischemia or reperfusion. Hearts perfused with standard buffer for an equal period of time served as controls. While the two groups demonstrated the same degree of postischemic increase in myocardial stiffness, peroxidase-pretreated hearts had a significantly better recovery of contractile indexes at 30 and 45 min of reflow. Our results confirm the ability of free-radical scavengers to enhance cardioplegic protection and point out the beneficial effects of peroxidase that, by inactivating both hydrogen peroxide and hydroperoxides, may prevent generation of the highly cytotoxic hydroxyl radical and interrupt the chain reaction of lipid peroxidation.