Adenosine has been shown to protect the ischemic and reperfused myocardium. To examine whether the protective effect of the nucleoside is mediated by modulation of oxidative stress, isolated rat hearts were perfused for 30 minutes with 100 μM H2O2or an exogenous free radical-generating system consisting of purine (3.06 mM) and xanthine oxidase (10 units/l) in the presence or absence of drugs acting on adenosine A1or A2receptors. H2O2alone produced a greater than 90% loss in contractility concomitant with a threefold elevation in resting tension, although these effects occurred in the absence of ultrastructural damage. Two A1receptor agonistsN6-cyclopentyladenosine (CPA, 1 μM) andR(−)-N6-(2-phenylisopropyl)adenosine (R-PIA, 1 μM) significantly attenuated the cardiodepressant effects of H2O2and depressed the elevation in resting tension; however, only the effect of CPA was found to be significant with regard to the latter parameter. A similar concentration ofS(+)-N6-(2-phenylisopropyl) adenosine (S-PIA), a markedly less potent A1receptor agonist, was found to be without beneficial effect. However, a significant protective effect against both the reduction in contractility and the elevation in resting tension was seen with a 10-fold elevation in the concentration of S-PIA (10 μM). The protective effects on functional parameters were associated with preservation of high-energy phosphate and adenine nucleotide contents after 30 minutes of H2O2treatment. The salutary effects of all drugs were reversed in the presence of the A1receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (0.5 μM). An A2receptor agonist 2-[p-(carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine, termed CGS 21680 (1 μM), failed to alter the cardiac response to H2O2with regard to all parameters studied. Neither a 50% reduction in external CaCl2concentration nor treatment with 10 μM dl-propranolol exerted salutary effects against H2O2-induced dysfunction. None of the A1receptor agonists modulated the response to purine plus xanthine oxidase. Our results demonstrate a selective protective effect of adenosine A1receptor activation against the cardiac toxicity of H2O2and provide, at least in part, a basis for the cardioprotective actions of adenosine and its analogues.