This study was designed to investigate whether a new irreversible injury to the myocardium can occur 6 hours after reperfusion following ischemia. Conscious rabbits were subjected to myocardial ischemia for 30 minutes, followed by reperfusion. Horseradish peroxidase (HPR) tracing with TTC staining and histological quantification were used to study the histopathological changes in myocardial tissues. Necrotic myocardium at 6 hours after reperfusion gave positive HRP results and viable myocardium at 24 hours after reperfusion gave positive TTC results. Some myocardial tissue between the above two regions were found to be HRP negative as well as TTC negative, indicating that myocardial tissue death occurred between 6 to 24 hours after reperfusion. The myocardial infarct size at 24, 48, and 72 hours after reperfusion were 7.0%, 5.1% and 3.2% larger than that 6, 24 and 48 hours respectively. Therefore, new irreversible myocardial injury in the rabbit can still occur after 6 hours of reperfusion following myocardial ischemia for 30 minutes and cumulative myocardial injury from 6 to 72 hours after reperfusion can result in a marked extension of the size of myocardial infarct.