Objectives: To characterize effects of A3 adenosine receptor (A3AR) activation and gene knock-out on responses to ischemia-reperfusion in mouse heart. Methods: Perfused hearts from wild-type and A3AR gene knock-out (A3AR KO) mice were subjected to 20 min ischemia and 30 min reperfusion. Functional responses were assessed and changes in energy metabolism and cytosolic pH monitored via 31P-NMR spectroscopy. Results: Selective A3AR agonism with 100 nM 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (chloro-IB-MECA) enhanced post-ischemic contractile recovery without altering contracture development in wild-type hearts, an effect unrelated to non-selective activation of A1 or A2 adenosine receptors. Chloro-IB-MECA also improved recovery in hearts overexpressing A1ARs. Paradoxically, post-ischemic recovery was enhanced by A3AR KO. Developed pressure, +dP/dt, and -dP/dt all recovered to higher levels in A3AR KO (70-80% of pre-ischemia) vs. wild-type hearts (45-50% of pre-ischemia) (P<0.05). Enhanced recovery was unrelated to recoveries of ATP, phosphocreatine (PCr), inorganic phosphate (Pi), energy state ([ATP]/[ADP]. [Pi], ?GATP) or cytosolic pH. Conclusions: Selective A3AR activation is cardioprotective in wild-type hearts and hearts overexpressing A1ARs, yet A3AR gene deletion generates an ischemia-tolerant phenotype without altering energy metabolism or pH. This may be due to compensatory changes or undefined genotypic differences in A3AR KO vs. wild-type hearts.