The left coronary artery was ligated and myocardial infarction developed in 28 rats. Three weeks later, the hearts were excised and mounted in an apparatus for perfusion of non-working isolated hearts (Langendorff). Hypothermic (15 degrees C), ischemic cardioplegia was induced for either 2 or 3 1/2 h followed by reperfusion for 45 min. Half of the hearts were reperfused with an initially gradual rise in temperature and pressure of the perfusion fluid, whereas the other half was reperfused directly with the perfusate at 37 degrees C and 100 cm H2O pressure. The hearts were examined by transmission electron microscopy and randomized for stereological analysis based on point counting on electron micrographs. Cardioplegia of 2 h duration was tolerated better than cardioplegia for 3 1/2 h (interstitial edema; P = 0.03, fraction of altered mitochondria; P = 0.001). Particularly in the hearts undergoing the longest cardioplegia, myocardial injury was less severe following a gentle reperfusion as compared with those exposed to the clinically common abrupt technique (fraction of mitochondria in the myocyte; P = 0.03, fraction of altered mitochondria; P = 0.008). In the interstitium, the luminal area of capillaries was significantly increased and the endothelial swelling less pronounced in the groups undergoing the gentle reperfusion technique, (luminal/endothelial fraction; P = 0.01). The study shows that previously infarcted hearts are susceptible to ischemic damage even after 2 h of regular hypothermic, ischemic cardioplegia and that a gentle reperfusion technique significantly ameliorates reperfusion injury.