Interventions such as glycogen depletion that limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus, it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether a novel glycogen phosphorylase inhibitor, ingliforib (CP-368,296), could reduce infarct size in both in vitro and in vivo rabbit models of ischemiareperfusion injury (30 min regional ischemia, followed by 120 min reperfusion). In Langendorff hearts, constant perfusion of ingliforib, started 30 min before regional ischemia, elicited a concentrationdependent reduction in infarct size;_- infarct size was reduced by 69% with 10 µM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest, anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg/kg/hr infusion) selected to achieve a free plasma concentration equivalent to an estimated EC₅₀ in the isolated hearts (1.2 µM, 0.55 µg/ml) significantly reduced infarct size by 50%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores preserved, in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate pressure product in the ingliforib group, although heart rate was modestly decreased post-ischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection, and treating diabetic patients at increased risk of cardiovascular disease.