Ischemic preconditioning with brief periods of ischemia followed by reperfusion protects the myocardium against a subsequent prolonged ischemic insult. Reperfusion may influence the protection given by ischemic preconditioning by washing out metabolites that are accumulated during the preconditioning ischemia. This study was designed to define the duration of reperfusion necessary to provide such protection. Hearts of anesthetized rats were preconditioned by occlusion of the left coronary artery for 5 minutes. This was followed by reperfusion for either 1 minute (n = 6) or 30 seconds (n = 6). The hearts were then subjected to a sustained occlusion of the left coronary artery for 45 minutes followed by reperfusion for 3 hours. Control (n = 11) hearts were subjected only to occlusion of the left coronary artery for 45 minutes followed by reperfusion for 3 hours. Infarct size was measured using tetrazolium and expressed as a percentage of the region at risk. After reperfusion for 1 minute there was a significant reduction in the size of the infarct (32.3 +/- 4.1%), expressed as a percentage of the zone at risk, when compared to controls (61.9 +/- 3.5%) (p < 0.01). However, the protection received by preconditioning was lost when reperfusion was limited to 30 seconds (infarct size 63.4 +/- 3.2%). The results show that the minimum period of reperfusion required to give protection after preconditioning ischemia lies between 30 seconds and 1 minute.