A protein kinase G (PKG) activator 8-(4-chlorophenylthio)-guanosine 3', 5'-cyclic monophosphate (CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for adenosine to initiate signaling from low-affinity A_2b receptors during early reperfusion thus allowing endogenous adenosine to activate survival kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether CPT might also sensitize A_2b receptors to adenosine. CPT (10 µM) during the first minutes of reperfusion markedly reduced infarction in isolated rabbit hearts undergoing 30-min regional ischemia/2-h reperfusion, and salvage was blocked by MRS1754, an A_2b-selective antagonist. Co-administration of wortmannin (PI3K inhibitor) or PD98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In non-ischemic hearts 10-min infusion of CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A_2b potent receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). However, when 2.5 nM NECA was combined with 10 µM CPT, both phospho-Akt and phospho-ERK1/2 significantly increased indicating CPT had lowered the threshold for A_2b-dependent signaling. The PKC antagonist chelerythrine blocked this phosphorylation induced by CPT+NECA. Chelerythrine also blocked CPT's anti-infarct effect as did non-selective (glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) K_ATP channel blockers. A free radical scavenger N-2-mercaptopropionyl glycine also blocked CPT's protection. We propose CPT targets PKG which activates PKC through mitoK_ATP-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac adenosine A_2b receptors so endogenous adenosine can protect by activating Akt and ERK.