We have previously reported that post-resuscitation myocardial dysfunction is accompanied by release of cytochrome c and caspase-3 activation. We now investigated the role of caspase-3 activation by examining whether such process prompts apoptotic DNA fragmentation, whether caspase-3 inhibition attenuates myocardial dysfunction, and whether myocardial protective effects of sodium-hydrogen exchanger isoform-1 (NHE-1) inhibition involve caspase-3 inhibition using a rat model of ventricular fibrillation (VF) of closed-chest resuscitation. Resuscitation after 4 or 8 minutes of untreated VF caused significant reductions in left ventricular stroke work index averaging 23% of sham control rats at 4 hours post-resuscitation. Left ventricular dysfunction was accompanied by increases in cytosolic cytochrome c, decreases in pro- and cleaved caspase-9 fragments, increases in 17-kDa caspase-3 fragments, and increases in caspase-3 activity indicating activation of the mitochondrial apoptotic pathway but without evidence of apoptotic DNA fragmentation. In addition, levels of HSP 70 were increased and levels of XIAP and crystallin were preserved, all of which can exert anti-apoptotic effects. In a separate series, the caspase-3 inhibitor z-DEVD-cmk given before induction of VF failed to prevent post-resuscitation myocardial dysfunction despite reductions in caspase-3 activity (2.3 ± 0.5 vs 1.3 ± 0.5 pmoles fluorophore AFC released/mg protein/min, p < 0.03). Treatment with the NHE-1 inhibitor cariporide had no effect on caspase-3 activity. Accordingly, in this rat model of VF and severe post-resuscitation myocardial dysfunction, activation of caspase-3 did not lead to DNA fragmentation or contributed to myocardial dysfunction. Concomitant activation of intrinsic anti-apoptotic mechanisms could play a protective role downstream to caspase-3 activation.