Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A(1) receptors (A(1)R). The role of PKCepsilon in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKCepsilon was overexpressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKCepsilon construct and compared with normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKCepsilon was found localized primarily in transverse tubules. The A(1)R agonist chlorocyclopentyladenosine (CCPA) at 1 microM rendered an enhanced localization of PKCepsilon in the t-tubular system. The beta-adrenergic agonist isoproterenol (Iso; 0.4 microM) elicited a 29-36% increase in myocyte shortening in all three groups. Although CCPA significantly reduced the Iso-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKCepsilon overexpression. The CCPA reduction of the Iso-elicited shortening was eliminated in the presence of a PKCepsilon inhibitory peptide. These results suggest that the translocation of PKCepsilon to the t-tubular system plays an important role in A(1)R-mediated antiadrenergic actions in the heart.