Myocardial protection associated with ischemic preconditioning (PC) wanes within an hour or two. It has recently been observed, however, that a delayed phase of protection appears about 24 h after ischemic PC in anesthetized rabbits and dogs which might be related to synthesis of cytoprotective proteins. We tested whether a second window of protection could be induced in conscious rabbits. Rabbits chronically instrumented with a coronary artery occluder and ECG electrodes experienced a 30-min coronary occlusion followed by 3 h reperfusion. Infarct size was measured with triphenyltetrazolium chloride. 35.7 +/- 2.3% of the risk zone infarcted in control animals. PC with 4 cycles of 5-min coronary occlusion/10-min reperfusion 24 h prior to the 30-min ischemia decreased infarction to 24.1 +/- 1.4% of the risk zone (P < 0.01). During the 30-min occlusion 3 of 7 non-PC rabbits developed ventricular fibrillation, while this arrhythmia did not occur in the 7 PC animals (P < 0.1). Myocardial hsp70 content in PC rabbits was twice that in controls. Collateral blood flow was not different in the two groups. A second window of protection exists in conscious rabbits which minimizes both infarction and arrhythmias, and cytoprotective protein content is increased in the myocardium of protected animals.