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Objectives: Both hypothermia and ischemic preconditioning are known to provide tolerance to myocardial ischemia and reperfusion. The aim of this study was to determine whether hypothermia during the ischemic preconditioning period attenuates the protective effect of ischemic preconditioning. Methods: Experiments were performed in buffer-perfused isolated rabbit hearts. All hearts underwent 45 minutes of regional ischemia, followed by 2 hours of reperfusion. Ischemic preconditioning was elicited by either one or four periods of 5 minutes of regional ischemia. Hypothermia (25° C) was induced beginning either 20 or 50 minutes before the 45-minute period of regional ischemia; normothermia (38° C) was restored 10 minutes before the 45-minute period of regional ischemia. Except for the hypothermic periods noted, hearts were maintained at 38° C. Results: Normothermic ischemic preconditioning with either one or four cycles of 5 minutes of coronary occlusion resulted in a profound reduction of infarct size (58% reduction with one cycle, p < 0.05; 95% reduction with four cycles, p < 0.01). Hypothermic ischemic preconditioning with one cycle of 5-minute coronary occlusion resulted in no reduction of infarct size but hypothermic ischemic preconditioning with four cycles of 5-minute coronary occlusions resulted in a 94% reduction of infarct size (p < 0.01). Myocardial glycogen and lactate levels were maintained near control levels during hypothermic ischemia. Conclusions: From these data we conclude that hypothermia during the preconditioning period increases the threshold for eliciting the infarct limitation of ischemic preconditioning. (J Thorac Cardiovasc Surg 1998;116:319-26)