Cardiac gap junction (GJ) channels, composed of connexins, allow electrical and metabolic couplings between cardiomyocytes, properties important for coordinated action of the heart as well as tissue homeostasis and control of growth and differentiation. Fibroblast growth factor-2 (FGF-2) is an endogenous growth-promoting protein, believed to participate in the short- and long-term responses of the heart to injury. We have examined short-term effects of FGF-2 on cardiac myocyte GJ–mediated metabolic coupling, using cultures of neonatal rat cardiomyocytes. FGF-2 decreased coupling between cardiomyocytes assessed by scrape dye loading as well as microinjection and dye transfer within 30 minutes of administration. Genistein blocked the effects of FGF-2. To determine the mechanism, we next assessed the effect of FGF-2 on expression, distribution, and phosphorylation of connexin43 (Cx43), which is a major cardiomyocyte connexin. FGF-2 did not affect Cx43 mRNA or protein accumulation and synthesis, and it did not change Cx43 localization at sites of intercellular contact as assessed by immunostaining with a polyclonal anti-Cx43 antibody raised against a synthetic peptide containing residues 346 to 363 of Cx43. FGF-2, however, decreased staining intensity at sites of intermyocyte contact when a monoclonal anti-Cx43 antibody was used, suggesting a localized masking of epitope(s) recognized by the monoclonal but not the polyclonal antibody. These epitopes appear to reside within residues 261 to 270 of Cx43, as indicated by full quenching of monoclonal antibody staining with synthetic peptides. In addition, FGF-2 induced a more than twofold increase in Cx43 phosphorylation. Phosphoamino acid analysis indicated increased phosphorylation of Cx43 on serine residues. Although tyrosine phosphorylation of Cx43 was not detected in either treated or control cells, a fraction of Cx43 was immunoprecipitated with anti–phosphotyrosine-specific antibodies in FGF-2–treated myocytes, suggesting interaction (and hence coprecipitation) with phosphotyrosine-containing protein(s). In conclusion, we have identified Cx43 and intercellular communication as targets of FGF-2–triggered and tyrosine phosphorylation–dependent signal transduction in cardiac myocytes. It is suggested that phosphorylation of Cx43 on serine induced by FGF-2 contributes to decreased metabolic coupling between cardiomyocytes.