AMP 579, an adenosine A₁/A₂ receptor agonist, has a strong anti-infarct effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether AMP 579 can reduce the production of reactive oxidant species (ROS) during reoxygenation in cultured chick embryonic cardiomyocytes. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH) was used to detect ROS. The cells were subjected to 60 min of simulated ischemia, followed by either 15 min or 3 h of reoxygenation. AMP 579 (0.5 and 1 µM), when started 10 min before reoxygenation, significantly reduced ROS generation from 4.86 ± 0.30 (arbitrary units) in untreated cells to 2.72 ± 0.31 and 1.85 ± 0.14, respectively (P < 0.05). Cell death that was assessed by propidium iodide uptake was markedly reduced by AMP 579 (49.6 ± 4.7% of control cells vs. 25.4 ± 2.4%, P < 0.05). In contrast, adenosine did not alter ROS generation or cell death. Attenuation of ROS production by AMP 579 was completely prevented by simultaneous exposure of cells to the selective adenosine A₂ antagonist 8-(13-chlorostyryl) caffeine. These results indicate that AMP 579 directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates intracellular oxidant stress. Furthermore, adenosine could not duplicate these effects.