The effect of adenosine on the increase in cardiac cyclic adenosine 3′,5′-monophosphate (cyclic AMP) concentration and glycogen phosphorylase activity produced by isoproterenol was investigated. Slices of rat ventricular myocardium 0.5 mm thick weighing 15-20 mg were cut, washed, and incubated at 37 °C in physiological saline gassed with either O2 (oxygenated) or N2 (anoxia). The concentration of cyclic AMP declined as the time of incubation increased in both oxygenated and anoxic muscle. In oxygenated muscle, isoproterenol, 10 fiM, produced a 2.2-fold increase in cyclic AMP concentration and phosphorylase activity. Adenosine at 1 fiM caused a 35% and 75% reduction in the isoproterenol-produced increase in cyclic AMP concentration and phosphorylase activity, respectively, without affecting basal levels. Reduction of the isoproterenol-elicited increase in cyclic AMP occurred within 2 minutes. Adenosine alone only at a high concentration of 1 nw increased cyclic AMP by 38% in oxygenated muscle. Adenine and inosine did not mimic the effect of adenosine on the isoproterenol-induced augmentation of cyclic AMP. Addition of adenosine deaminase to the physiological saline prevented the effects of adenosine but did not affect basal cyclic AMP. In anoxic tissue, isoproterenol failed to produce an increase in cyclic AMP and phosphorylase. Addition of adenosine deaminase to anoxic tissue resulted in an isoproterenol-produced increase in cyclic AMP, indicating that adenosine may inhibit an isoproterenol-induced increase in cyclic AMP during anoxia. These results suggest that adenosine attenuates the catecholamine-induced increase in cyclic AMP concentration and phosphorylase activity in oxygenated cardiac muscle, whereas, in the anoxic myocardium, adenosine may be responsible for preventing an increase in cyclic AMP upon β-adrenergic stimulation. Thus, adenosine may antagonize catecholamine elicited glycogenolysis.