It has been proposed that the formation of gap junction is influenced by adherens junction in cardiac myocytes. To examine whether signals through N-cadherin are involved in the distribution of connexin 43 (Cx43), the distribution of cell–cell adhesion molecules, N-cadherin and Cx43, was analyzed in aligned cardiac myocytes. To induce cell orientation running in parallel to tension direction, neonatal rat cardiac myocytes were plated for 3 hours and exposed to 20% cyclic stretch for 24 hours on silicone dishes. The aligned cells cultured for 0–5 days were immunostained with anti- N-cadherin or anti-Cx43 antibody. After cultivation for 3–5 days, following the accumulation of N-cadherin, Cx43 was localized at the longitudinal cell termini. Adenoviral gene transfer of dominant negative N-cadherin significantly attenuated the localization of Cx43 at the longitudinal cell termini, suggesting that Cx43 localization is regulated downstream of N-cadherin. In the process of Cx43 localization, Rho family proteins, RhoA and Rac1, were activated, but not Cdc42. RhoA and Rac1 activation was inhibited by the transfection of dominant negative N-cadherin, indicating that RhoA and Rac1 were activated by N-cadherin in the oriented cardiac myocytes. The inhibition of Rho family proteins by Rho GDI significantly attenuated the accumulation of Cx43, but not that of N-cadherin. Furthermore, the translocation of Cx43 to longitudinal cell termini was prevented by the inhibition of Rac1, but not RhoA. Collectively, these findings suggest that the localization of Cx43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes.