Reperfusion-induced calcium gain provides a marker of irreversible injury, but whether the cells gain calcium because of irreversible injury caused by the ischemic episode, or whether it is the reperfusion-induced calcium gain that triggers the irreversible injury has yet to be established. Using isolated rat hearts made ischemic for either 30 or 60 minutes, and reperfusing with Krebs-Henseleit buffer or Krebs-Henseleit buffer containing either 2,3-butanedione monoxime (to inhibit contractile activity) or 2,4-dinitrophenol or nitrogen-gassed substrate-free Krebs-Henseleit buffer (to inhibit oxidative phosphorylation), the effect of reperfusion was monitored in terms of calcium gain and ultrastructural changes including loss of sarcolemmal integrity. The results establish that the routes of calcium entry during postischemic reperfusion are complex. The calcium gain can occur in the absence of mitochondrial oxidative phosphorylation and is modulated by interventions introduced at the moment of reperfusion which affect the contractile state. There are at least 2 routes of calcium entry: contraction-dependent and contraction independent. The former is probably associated with the development of sarcolemmal discontinuities. The results also establish that when sarcolemmal integrity has been destroyed, the cells can gain excess calcium under conditions that prevent mitochondrial calcium uptake. Accordingly, the mitochondria cannot be the only intracellular organelles that accumulate calcium under these conditions. Additional studies are needed to identify the other sites of calcium binding under conditions of adenosine triphosphate deprivation.