The influence of the adenosine derivatives (−)-N⁶-phenylisopropyladenosine (R-PIA, 1 μm) and 5′-N-ethylcarbox-amidoadenosine (NECA, 1 μm) on β-adrenergic stimulated (isoproterenol, 10 nm) phosphorylation of sarcolemmal (15 kDa protein), sarcoplasmic reticular (phospholamban) and myofibrillar proteins (troponin I. C-protein) was studied in isolated ³²P-labeled guinea-pig ventricles. The identification of the 15 kDa protein, phospholamban, troponin I and C-protein was based on their reaction with specific antibodies. Isoproterenol increased contractile parameters (developed tension, rate of tension development, rate of relaxation) and stimulated the phosphorylation state of a 15 kDa protein (now named phospholemman), of phospholamban, troponin I and C-protein (regarded as regulatory proteins). Isoproterenol concomitantly increased myocardial cyclic AMP levels. R-PIA and NECA attenuated the effects of isoproterenol on contractile parameters as well as on the phosphorylation of the regulatory proteins without affecting cyclic AMP levels. The effects of 1 μm R-PIA and 1 μm NECA on the isoproterenol-stimulated phosporylation of regulatory proteins were blocked by the adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 μm). Therefore, it is concluded that adenosine derivatives acting via adenosine receptors can reduce the isoproterenol-stimulated phosphorylation state of the following regulatory proteins: phospholemman, phospholamban, troponin I and C-protein.