Eph receptors and ephrin ligands are membrane-bound cell–cell communication molecules with important roles not only in development but also in the physiology of many adult organs. However, their cellular localization and functions in the myocardium are virtually unknown and therefore, we have investigated the expression of EphB receptors and ephrin-B ligands in the rodent heart ventricles and their functions in the rodent cardiomyocytes of primary culture. Examinations by RT-PCR, immunohistochemistry and in situ hybridization revealed that the EphB receptors are preferentially expressed in cardiomyocytes and ephrin-B ligands in the vasculature in adult mouse heart ventricles. Interestingly, we found that inducing high levels of EphB receptor activation in primary cultures of rodent cardiomyocytes by stimulation with ephrin-B1-Fc desynchronized the contraction of adjacent clusters of cardiomyocytes that had contracted synchronously before the treatment. Co-immunoprecipitation experiments revealed that EphB4 physically associates with connexin43, a major component of gap junctions in the myocardium, and that EphB activation inhibits gap junctional intracellular communication between cardiomyocytes. The present findings suggest that ephrin-B-EphB signaling can modulate the electrical coupling of cardiomyocytes through effects on gap junctions.