We recently demonstrated that after 1 hr of simulated ischemia, reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes. Here, we tested whether ischemia/reperfusion-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels by 1 hr of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 ± 0.52 - fold increase (over ischemia only) within the first hour of reperfusion, while the activities of caspases-3, -8 and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor (z- VDVAD-fmk) was the only caspase inhibitor that significantly inhibited cyto c release from mitochondria. This inhibitor also completely blocked the activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked the activity of caspase-2 and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block either caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release following reperfusion, and that it is critical for I/R-induced apoptosis seen in this model.