Adenosine-induced antiadrenergic effects in the heart are mediated by adenosine A₁ receptors (A₁R). The role of protein kinase C epsilon (PKC) in the antiadrenergic action of adenosine was explored with adult rat ventricular myocytes in which PKC was over-expressed. Myocytes were transfected with a pEGFP-N1 vector in the presence or absence of a PKC construct and compared to normal myocytes. The extent of myocyte shortening elicited by electrical stimulation of quiescent normal and transfected myocytes was recorded with video imaging. PKC was found localized primarily in transverse tubules. The A₁R agonist chlorocyclopentyladenosine (CCPA) at 1 µM rendered an enhanced localization of the PKC in the t-tubular system. The - adrenergic agonist isoproterenol (ISO, 0.4 µM) elicited a 29-36% increase in myocyte shortening in all three groups. While CCPA significantly reduced the ISO-produced increase in shortening in all three groups, the reduction caused by CCPA was greatest with PKC over-expression. The CCPA reduction of the ISO-elicited shortening was eliminated in the presence of a PKC inhibitory peptide. These results suggest that the translocation of PKC to the t-tubular system plays an important role in A₁R-mediated antiadrenergic actions in the heart.