Gap junctions are intercellular communicating channels responsible for the synchronized activity of cardiomyocytes. Recent studies have shown that the membrane-associated guanylate kinase protein, zonula occludens-1 (ZO-1) can bind to catenins in epithelial cells and act as an adapter for the transport of the connexin isotype, Cx43 during gap junction formation. The significance of catenins in the development of gap junctions and whether complexes between catenins and ZO-1 are formed in cardiomyocytes are not clear. In this study, immunofluorescence and confocal microscopy showed sequential redistribution of α-catenin, β-catenin, ZO-1, and Cx43 to the plasma membrane when rat cardiomyocytes were cultured in low Ca2+ (<5 μM) medium, then shifted to 1.8 mM Ca2+ medium (Ca2+ switch). Diffuse cytoplasmic staining of α-catenin, α-catenin, ZO-1, and Cx43 was seen in the cytoplasm when cardiomyocytes were cultured in low Ca2+ medium. Staining of α-catenin, β-catenin, and ZO-1 was detected at the plasma membrane of cell - cell contact sites 10 min after Ca2+ switch, whereas Cx43 staining was first detected, colocalized with ZO-1 at the plasma membrane, 30 min after Ca2+ switch. Distinct junctional and extensive cytoplasmic staining of α-catenin, β-catenin, ZO-1, and Cx43 was seen 2 h after Ca2+ switch. Immunoprecipitation of Triton X-100 cardiomyocyte extracts using anti-β-catenin antibodies showed that β-catenin was associated with β-catenin, ZO-1, and Cx43 at 2 h after Ca2+ switch. Intracellular application of antisera against α-catenin, β-catenin, or ZO-1 by electroporation of cardiomyocytes cultured in low Ca2+ medium inhibited the redistribution of Cx43 to the plasma membrane following Ca2+ switch. These results suggest the formation of a catenin-ZO-1-Cx43 complex in rat cardiomyocytes and that binding of catenins to ZO-1 is required for Cx43 transport to the plasma membrane during the assembly of gap junctions.