Post-ischemic myocardial dysfunction has been observed in a variety of clinical situations including cardiac arrest. Potentially survivable cardiac arrest following short-term global myocardial ischemia may be of insufficient duration to cause irreversible myocyte injury, but still results in contractile and bioenergetic dysfunction. The purpose of this study was to characterize the ischemic transition from reversible to irreversible injury in the isolated perfused rat heart. Isolated, buffer perfused, male Sprague-Dawley rat hearts underwent normothermic ischemia of 15, 20, 25 or 30 min with or without 30 min of reperfusion and were freeze clamped in liquid nitrogen for bioenergetic analysis of LV tissue. Post-ischemic LV function and measurements of bioenergetic recovery were made between groups and with non-ischemic controls. Baseline LV function was similar in all groups. Post-ischemic contractile function was markedly depressed in the 25 and 30 min ischemia groups with persistent depression of high-energy phosphates, total adenine nucleotide pool, myocardial oxygen consumption, elevated CK release and evidence of significant mitochondrial edema in the 30 min group. In contrast with longer ischemic periods, the reduction in LV contractile function after 15 and 20 min of ischemia was mild, with more complete bioenergetic recovery, minimal CK release, and normal appearing mitochondrial. This data suggests a period of transition from reversible to irreversible injury occurring at approximately 20 min of normothermic global ischemia in the isolated perfused rat heart.