It has been recently shown that beta-adrenergic receptors are able to activate phospholipase C via the cyclic adenosine monophosphate-binding protein Epac. This new interconnection may participate in isoproterenol (Iso)-induced preconditioning. We evaluated here whether Epac could induce PKCepsilon activation and could play a role in ischemic preconditioning through the phosphorylation of connexin43 (Cx43) and changes in gap junctional intercellular communication (GJIC). In cultured rat neonatal cardiomyocytes, we showed that in response to Iso and 8-CPT, a specific Epac activator, PKCepsilon content was increased in particulate fractions of cell lysates independently of protein kinase A (PKA). This was associated with an increased Cx43 phosphorylation. Both Iso and 8-CPT induced an increase in GJIC that was blocked by the PKC inhibitor bisindolylmaleimide. Interestingly, inhibition of PKA partly suppressed both Iso-induced increases in Cx43 phosphorylation and in GJIC. The same PKCepsilon-dependent Cx43 phosphorylation by beta-adrenergic stimulation via Epac was found in adult rat hearts. However, in contrast with Iso that induced a preconditioning effect, perfusion of isolated hearts with 8-CPT prior to ischemia failed to improve the post-ischemia functional recovery. In conclusion, Epac stimulation induces PKCepsilon activation and Cx43 phosphorylation with an increase in GJIC, but Epac activation does not induce preconditioning to ischemia in contrast with beta-adrenergic stimulation.