Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 ± 3.8%; 5 μM fasudil, 18.2 ± 3.1%; 0.3 μM Y-27632, 19.4 ± 4.4%; 5 μM Y-27632, 9.2 ± 2.9%). When 5 μM Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 ± 2.6% vs. control 33.4 ± 4.4%, P < 0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 ± 6.1%) and N^ω-nitro-l-arginine methyl ester (30.4 ± 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, suggesting that ROCK activation at reperfusion may be deleterious through suppression of the RISK pathway.