Activation of A₁ adenosine receptors (A₁ARs) may be a crucial step in protection against myocardial ischemia-reperfusion (I/R) injury; however, the use of pharmacological A₁AR antagonists to inhibit myocardial protection has yielded inconclusive results. In the current study, we have used mice with genetically modified A₁AR expression to define the role of A₁AR in intrinsic protection and ischemic preconditioning (IPC) against I/R injury. Normal wild-type (WT) mice, knockout mice with deleted (A₁KO^−/−) or single-copy (A₁KO^+/−) A₁AR, and transgenic mice (A₁TG) with increased cardiac A₁AR expression underwent 45 min of left anterior descending coronary artery occlusion, followed by 60 min of reperfusion. Subsets of each group were preconditioned with short durations of ischemia (3 cycles of 5 min of occlusion and 5 min of reperfusion) before index ischemia. Infarct size (IF) in WT, A₁KO^+/−, and A₁KO^−/− mice was (in % of risk region) 58 ± 3, 60 ± 4, and 61 ± 2, respectively, and was less in A₁TG mice (39 ± 4, P < 0.05). A strong correlation was observed between A₁AR expression level and response to IPC. IF was significantly reduced by IPC in WT mice (35 ± 3, P < 0.05 vs. WT), A₁KO^+/− + IPC (48 ± 4, P < 0.05 vs. A₁KO^+/−), and A₁TG + IPC mice (24 ± 2, P < 0.05 vs. A₁TG). However, IPC did not decrease IF in A₁KO^−/− + IPC mice (63 ± 2). In addition, A₁KO^−/− hearts subjected to global I/R injury demonstrated diminished recovery of developed pressure and diastolic function compared with WT controls. These findings demonstrate that A₁ARs are critical for protection from myocardial I/R injury and that cardioprotection with IPC is relative to the level of A₁AR gene expression.