—Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the ε-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2–induced Cx43 phosphorylation. In nonstimulated myocytes, PKCε displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCε distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCε. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCε but not PKCα coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCε, suggesting increased association between PKCε and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCε decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2–induced effects on cardiac GJs and that PKCε likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.