The purpose of this study was to determine whether the protective effects of adenosine on myocardial ischemia-reperfusion injury are altered with age, and if so, to clarify the mechanisms that underlie this change related to nitric oxide (NO) derived from the vascular endothelium. Isolated perfused rat hearts were exposed to 30 min of ischemia and 60 min of reperfusion. In the adult hearts, administration of adenosine (5 μmol/l) stimulated NO release (1.06 ± 0.19 nmol · min−1 · g−1, P < 0.01 vs. vehicle), increased coronary flow, improved cardiac functional recovery (left ventricular developed pressure 79 ± 3.8 vs. 57 ± 3.1 mmHg in vehicle, P < 0.001; maximal rate of left ventricular pressure development 2,385 ± 103 vs. 1,780 ± 96 in vehicle, P < 0.001), and reduced myocardial creatine kinase loss (95 ± 3.9 vs. 159 ± 4.6 U/100 mg protein, P < 0.01). In aged hearts, adenosine-stimulated NO release was markedly reduced (+0.42 ± 0.12 nmol · min−1 · g−1 vs. vehicle), and the cardioprotective effects of adenosine were also attenuated. Inhibition of NO production in the adult hearts significantly decreased the cardioprotective effects of adenosine, whereas supplementation of NO in the aged hearts significantly enhanced the cardioprotective effects of adenosine. The results show that the protective effects of adenosine on myocardial ischemia-reperfusion injury are markedly diminished in aged animals, and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration.