Experiments were carried out in hearts from guinea pigs that were fed either the adenosine receptor antagonist theophylline (0.6 mg/ml) or no drug. The Aiadenosine receptor radioligand [125l]aminobenzyladenosine bound to a single affinity class of receptors in heart cell membranes from control animals with Bmaxand KDof 183 ± 1.0 fmol/mg protein and 3.7 ± 0.6 nM, respectively (n=8). Heart cell membranes from animals fed theophylline for 2, 7, and 14 days bound the radioligand with about the same affinity, but the number of binding sites was significantly increased (p<0.01) to 30.6 ± 1.7 (n=3), 30.0 ± 0.8 (n=3), and 27.3 ± 2.9 (n=4), respectively. Nearly identical results were obtained with membranes prepared from enzymatically dispersed ventricular myocytes. Fourteen days of theophylline treatment also produced a small increase (12%,p< 0.01) in the number of binding sites in membranes derived from cerebral cortexes. Isolated ventricular myocytes prepared from animals fed no drug or theophylline for 7 days were used to determine the effect of adenosine on 20 nM isoproterenolstimulated calcium current (Icameasured by the whole-cell patch-clamp technique. Adenosine reduced isoproterenol-stimulated Icawithout affecting the activation or inactivation kinetics of the current; Icadensity was reduced less by 5 μM adenosine in cells from control (25 ± 3 to 21 ± 3 μA/μF) than in cells from theophylline-fed animals (26 ± 5 to 17 ± 2 μA/μF). Although a high concentration (0.5 mM) of adenosine abolished isoproterenol-stimulated Icain cells from control or theophylline-fed animals, the IC50for adenosine was sixfold less in cells derived from theophylline-fed animals than in cells from control animals (4.6 ± 0.6 μM and 283 ± 1.4 μM, respectively,p< 0.01). In contrast, the increase in Icain response to isoproterenol alone and the potency of acetylcholine to antagonize this effect of isoproterenol were the same in both groups of cells. A maximally effective concentration ofR-phenylisopropyladenosine (0.1 mM) inhibited isoproterenol-stimulated cyclic AMP accumulation less in cardiomyocytes from control than from theophylline-fed animals (28.7 ± 1.8% vs. 42.0 ± 4.2%,p< 0.05). In summary, exposure of the myocardium to theophylline increases the number of adenosine receptors and the effects of receptor occupancy by agonists. These findings imply that the endogenous concentration of adenosine is high enough in the normoxic guinea pig heart to chronically maintain adenosine receptors in a partially down-regulated state.