Although protein kinase C (PKC) and phosphatidylinositol 3 (PI3)-kinase are implicated in cardioprotective signal transduction mediated by ischemic preconditioning, their role in pharmacological preconditioning (PPC) has not been determined. Cultured neonatal rat cardiomyocytes (CMCs) were subjected to simulated ischemia for 2 hours followed by 15 minutes reoxygenation. CMCs underwent PPC with 50 µM adenosine, 50 µM diazoxide, and 50 µM S-nitroso-N-acetyl-penicillamine (SNAP) each alone or in combination for 15 minutes followed by 30 minutes washout before simulated ischemia. Although PKC- and PI3-kinase were significantly activated during treatment with adenosine, activation of these kinases was dissipated after washout. In contrast, combined PPC with adenosine, diazoxide, and SNAP elicited sustained activation of PKC- and PI-3 kinase after washout. The combined, but not single PPC protocol conferred anti-apoptotic and anti-necrotic effects after reoxygenation The PKC inhibitor chelerythrine (5 µM) or the PI-3 kinase inhibitor LY294002 (10 µM) given during the washout period partially blocked the activation of PKC- and PI-3 kinase mediated by the combined PPC, while combined addition of chelerythrine and LY294002 completely inhibited the activation of PKC- and PI-3 kinase. Chelerythrine or LY294002 partially blocked anti-apoptotic and anti-necrotic effects mediated by the combined PPC, while combined addition of chelerythrine and LY294002 completely abrogated anti-apoptotic and anti-necrotic effects. These results suggest that combined PPC confers cardioprotective memory through sustained and interdependent activation of PKC and PI3-kinase.