Ultrastructural changes in cardiac muscle of isolated working rabbit hearts after various periods of ischemia are described and compared with distributional changes in calcium. The effects of reperfusion on these structural parameters were also investigated. The purposes of this study were to relate the role of calcium in the degeneration of cardiac muscle; to determine whether Ca2+ localizations could serve as additional criteria to determine more closely the point of no return; and to investigate the contributory role of reoxygenation to the development of myocardial damage. This study shows the existence of topographic differences in the tolerance to ischemia in the mid area, subendocardium, and subepicardium; that the sequestration of Ca2+ by mitochondria is an energy-requiring (active) process that occurs only during reperfusion; the loss of the sarcolemma's ability to bind Ca2+ during ischemia to coincide with increased Ca2+ entry during postischemic reperfusion (this Ca2+ is scavenged by mitochondria as long as sufficient energy remains available; these changes are interpreted as being at the edge of irreversibility); and the lack of additional damage and and lack of Ca2+ accumulation in mitochondria during reperfusion in cells that are damaged to such an extent that mitochondria possess flocculent densities already at the end of the ischemic insult.