Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A(1) receptors (A(1)R) on the adrenergic signaling pathway. An indirect A(1)R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-epsilon to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A(1)R activation of PKC-epsilon induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A(1)R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-epsilon and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-epsilon to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A(1)R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-epsilon co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-epsilon with RACK2 by 61%. With rat cardiomyocytes, the beta-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A(1)R stimulation of the heart is associated with PLC-initiated PKC-epsilon translocation and association with RACK2.