Pervious biochemical and hemodynamic studies have highlighted the important role of εPKC in cardioprotection during ischemic preconditioning. However, little is known about the electrophysiological consequences of εPKC modulation in ischemic hearts. Membrane permeable peptide εPKC selective activator and inhibitor were used to investigate the role of εPKC modulation in reperfusion arrhythmias. Methods: Protein transduction domain from HIV-TAT was used as a carrier for peptide delivery into intact Langendorff perfused guinea pig hearts. Action potentials were imaged and mapped (124 sites) using optical techniques and surface ECG was continuously recorded. Hearts were exposed to 30min stabilization period, 15min of no-flow ischemia, followed by 20min reperfusion. Peptides (0.5μM) were infused as follows: (a) control (vehicle-TAT peptide; TAT-scrambled ψεRACK peptide); (b) εPKC agonist (TAT-ψεRACK); (c) εPKC antagonist (TAT-εV1). Results: Hearts treated with εPKC agonist ψεRACK had reduced incidence of ventricular tachycardia (VT, 64%) and fibrillation (VF, 50%) compared to control (VT, 80%, P<0.05) and (VF, 70%, P<0.05). However, the highest incidence of VT (100%, P<0.05) and VF (80%) occurred in hearts treated with εPKC antagonist peptide εV1 compared to control and to εPKC agonist ψεRACK. Interestingly, at 20min reperfusion, 100% of hearts treated with εPKC agonist ψεRACK exhibited complete recovery of action potentials compared to 40% (P<0.05) of hearts treated with εPKC antagonist peptide, εV1 and 65% (P<0.5) of hearts in control. At 20min reperfusion, maps of action potential duration from εPKC agonist ψεRACK showed minimal dispersion (48.2±9ms) compared to exacerbated dispersion (115.4±42ms, P<0.05) in εPKC antagonist and control (67±20ms, P<0.05). VT/VF and dispersion from hearts treated with scrambled agonist or antagonist peptides were similar to control. Conclusion: The results demonstrate that εPKC activation by ψεRACK peptide protects intact hearts from reperfusion arrhythmias and affords better recovery. On the other hand, inhibition of εPKC increased the incidence of arrhythmias and worsened recovery compared to controls. The results carry significant therapeutic implications for the treatment of acute ischemic heart disease by preconditioning-mimicking agents.