Background: Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here, we investigate the role of ROCK activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway PI3k/Akt/eNOS. Methods: Rat isolated hearts underwent 35 min left coronary artery occlusion and 120 min 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 TUNEL positivity was used as an index of apoptosis. Results: The ROCK inhibitors fasudil or Y27632 given 10 min prior to ischemia until 10 min after reperfusion reduced infarct size (control 34.1±3.8%; fasudil 5µM 18.2 ± 3.1%; Y27632 0.3 µM 19.4±4.4%; Y27632 5 µM 9.2±2.9%). When Y27632 5 µM 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 Y27632 given at reperfusion was attenuated by wortmannin (29.2±6.1%) and L-NAME (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 Y27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL positive nuclei in the infarcted and peri-infarct regions. Conclusions: 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.