Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 ± 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 ± 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G_i protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.