To date, the involvement of reactive oxygen species in ischemic preconditioning in vivo in rats is not clearly demonstrated. The aim of the present study was to determine whether N-(2-mercaptopropionyl)glycine (MPG), a cell-diffusible hydroxyl radical scavenger, and carnosine, a potent singlet oxygen quencher, could block protection afforded by a single cycle of ischemic preconditioning in vivo in the rat. An ESR study was first performed to validate in vitro the specific antioxidant properties of carnosine and MPG. In a second set of experiments, open-chest rats were subjected to 30 min of left coronary occlusion followed by 60 min of reperfusion. Preconditioning was elicited by 5 min of ischemia and 5 min of reperfusion. Neither MPG (1-h infusion, 20 mg/kg) nor carnosine injection (bolus, 25 micro mol/rat) affected infarct size. The infarct size-limiting effect of preconditioning was completely blunted by MPG, whereas carnosine did not alter the cardioprotection. It is concluded that free radicals and especially hydroxyl radicals could be involved in the adaptive mechanisms induced by a single cycle of preconditioning in vivo in rats.