To determine whether A(3) adenosine receptor (A(3)AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A(3)AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while (31)P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A(3)AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A(3)AR-hearts. To determine the role of depressed heart rate and to confirm A(3)AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A(3)AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A(3)AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A(3)AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A(3)AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A(3)AR overexpression results in cardioprotection via a specific A(3)AR effect, possibly involving preservation of ATP during ischemia.