Isolated guinea pig hearts were used to determine whether an extracellular (interstitial) or intracellular pool of myocardial adenosine is most important in attenuating the catecholamine-induced enhancement of cardiac contractile state and glycogenolysis. Isoproterenol (2 X 10(-8) M) stimulation of hypoxic (30% O2) perfused hearts produced a marked elevation in tissue and effluent perfusate adenosine levels that were greater than the increases observed with the isoproterenol stimulation of oxygenated hearts (95% O2). In the isoproterenol stimulated hypoxic hearts nitrobenzylthioinosine (NBMPR), a potent inhibitor of adenosine cellular transport, further increased tissue adenosine content and markedly decreased the perfusate level of the nucleoside. Assuming that perfusate levels of adenosine correlate directly with extracellular levels, NBMPR was used as a tool to increase the intracellular and decrease the extracellular content of the nucleoside. When compared to responses in oxygenated hearts, hypoxia reduced the isoproterenol-produced increase in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractility but enhanced the increase in glycogen phosphorylase alpha formation. NBMPR completely prevented the reduction of the isoproterenol-induced cyclic AMP and cyclic AMP-dependent protein kinase responses but only partially prevented the attenuation of the contractile response. The increase in phosphorylase alpha formation in the hypoxic isoproterenol stimulated hearts was not influenced by NBMPR. The results suggest that an increase in extracellular adenosine is more influential than an elevation of intracellular adenosine in attenuating beta-adrenoceptor-elicited increases in myocardial cyclic AMP content, cyclic AMP-dependent protein kinase activity and contractile state.