We aimed to test if stimulation of both adenosine A2A and A2B receptors is required to produce an effective cardioprotection against reperfusion injury. Isolated rat hearts were subjected to 30-min regional ischemia followed by 2 h of reperfusion. The adenosine A1/A2 receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size, an effect that was reversed by both the adenosine A2A antagonist SCH58261 and the A2B antagonist MRS1706. The A2B agonist BAY 60-6583 but not the selective A2A agonist CGS21680 reduced infarct size. Interestingly, a combination of BAY 60-6583 and CGS21680 further reduced infarct size. These results suggest that both A2A and A2B receptors are involved in NECA's anti-infarct effect at reperfusion. NECA attenuated mitochondrial swelling upon reperfusion and this was blocked by both SCH58261 and MRS1706, indicating that activation of A2 receptors with NECA can modulate reperfusion-induced mitochondrial permeability transition pore (mPTP) opening. In support, NECA also prevented oxidant-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and matrix Ca2+ overload in cardiomyocytes via both the A2 receptors. In addition, NECA increased mitochondrial glycogen synthase kinase-3beta (GSK-3beta) phosphorylation upon reperfusion and this was again blocked by SCH58261 and MRS1706. In conclusion, A2A and A2B receptors work in concert to prevent reperfusion injury in rat hearts treated with NECA. NECA may protect the heart by modulating the mPTP opening through inactivating mitochondrial GSK-3beta. A simultaneous stimulation of A2A and A2B receptors at reperfusion is required to produce a strong cardioprotection against reperfusion injury.