Irreversible ischemic injury occurs after coronary artery occlusion in vivo, first in the subendocardium and progressing toward the subepicardium over time, presumably due to transmural variations in collateral flow or wall tension. In this study, 10 left ventricular globally ischemic slabs were created that were free of wall tension and collateral flow. The onset and completion of ischemic contracture were identified by means of a new tissue compressibility gauge designed for these studies. Transmural samples were obtained at 15 min intervals for determination of high-energy nucleotide levels and for ultrastructural analysis. The results show that there is a statistically significant gradient of ATP depletion, with the subendocardium consistently accelerated energy utilization compared with the subepicardium (p < .05). Ultrastructural evidence of irreversible injury first appeared in the subendocardium at the onset of ischemic contracture and occurred when ATP levels declined to less than 1 μmol/g wet weight. In summary, these data show that during total ischemia in vitro, cell death begins in the subendocardium at the onset of ischemic contracture and progresses toward the subepicardium over time. These changes occurred independent of variations in collateral flow or wall tension. The results suggest that the increased risk of the subendocardium to ischemic injury previously noted in vivo may occur not only because of variations in collateral flow and wall tension, but may also be secondary to an increased metabolic rate of the subendocardium resulting in faster ATP use during the period of ischemia.