Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A₁ receptor (A₁R). Since ₁-adrenergic receptor (₁R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the ₁R adrenergic and A₁R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the ₁R agonist isoproterenol (ISO) and/or the A₁R agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA). ³²P incorporation into protein substrates of 140-120,43 and 29 kD with PKA increased both the ISO-elicited activation of AC by 51-54% and the A₁R-mediated reduction of the ISO-induced increase in AC by 29-50% yielding a total antiadrenergic effect of ~78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of solubilized membrane adenylyl cyclase using a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by it's antiadrenergic action is facilitated by cardiac membrane phosphorylation.