The ability of adenosine A₁ receptors to activate type 2a protein phosphatase (PP2a) and account for antiadrenergic effects was investigated in rat myocardial preparations. We observed that the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) significantly reduces the isoproterenol-induced increase in left ventricular developed pressure of isolated heats, and this effect is blocked by pretreatment of hearts with the PP2a inhibitor cantharidin. CPA alone or given in conjunction with isoproterenol stimulation decreases phosphorylation of phospholamban and troponin I in ventricular myocytes. These dephosphorylations are blocked by an adenosine A₁ receptor antagonist and by PP2a inhibition with okadaic acid. Adenosine A₁ receptor activation was also shown to increase carboxymethylation of the PP2a catalytic subunit (PP2a-C) and cause translocation of PP2a-C to the particulate fraction in ventricular myocytes. These results support the hypothesis that adenosine A₁ receptor activation leads to methylation of PP2a-C and subsequent translocation of the PP2a holoenzyme. Increases in localized PP2a activity lead to dephosphorylation of key cardiac proteins responsible for the positive inotropic effects of -adrenergic stimulation.