Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct antiadrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging and co-immunoprecipitation (co-IP) whether A1R activation of PKC induces the kinase translocation to RACK2 in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC and RACK2 antibodies with secondaries conjugated to Cy3- and Cy5-(indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC to reversibly co-localize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine (PIA) to activate A1R, or with phorbol 12-myristate 13-acetate (Pb) to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, PIA and Pb in the rat heart increased the PKC co-IP with RACK2 by 186%, 49% and >1000%, respectively. The A1R antagonist DPCPX prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC with RACK2 by 61%. With rat cardiomyocytes, the -adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and DPCPX. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC translocation and association with RACK2.