Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since activation of A_2A adenosine receptors (A_2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from 5 groups of congenic mice: A_2AAR^+/+; A_2AAR^-/-; mast cell deficient (KitW-sh/W-sh); and chimeric mice prepared by transplanting bone marrow from A_2AAR^-/- or A_2AAR^+/+ mice to radiation-ablated A_2AAR^+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated-perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS21680 (100 nmol/L) decreased infarct size (IS, % area at risk) from 38 ± 2% to 24 ± 2% and 22 ± 2% in ATL146e- and CGS21680-treated hearts, respectively (P<0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A_2AAR^-/- hearts and in hearts from chimeric mice with A2AAR-/- bone marrow. Vehicle-treated KitW-sh/W-sh mice had lower IS (11 ± 3%) than WT mice, and ATL146e had no significant protective effect (16 ± 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A_2AAR activation is cardioprotective in the isolated heart at least in part by attenuating resident mast cell degranulation.