Using an in vitro system for differentiation of embryonic stem (ES) cells into cardiac myocytes, we analyzed the expression of connexin (Cx) genes by RT-PCR to learn what changes in the expression of multiple connexin genes occur during the early stage of heart development. We also examined gap junctional intercellular communication by using Lucifer Yellow dye microinjection transfer, studied intracellular Ca2+ transients by confocal laser image analysis using fluo 3, and determined localization of Cx43 by immunofluorescence during in vitro differentiation of ES cells into cardiac myocytes. The transcripts for Cx43 and Cx45 were detected in undifferentiated ES cells and in embryoid bodies before and after the appearance of beating cardiomyocytes. In contrast, Cx40 transcripts were not observed in undifferentiated ES cells and were barely detectable in 3- and 5-day-old embryoid bodies. Cx40 transcripts significantly increased with the appearance of beating cells similar to those of cardiac-specific genes. Dye coupling was present among undifferentiated ES cells, prebeating cells of embryoid body outgrowth and ES cell-derived beating cardiomyocytes. When dye was injected into a beating cell, dye spread was restricted to neighboring beating cells. Immunofluorescence demonstrated that Cx43 protein was localized not only in beating cells but also in surrounding nonbeating cells, whereas myosin heavy chain alpha/beta was exclusively positive in the beating cells. These data suggest that the expression of multiple connexins is differentially regulated during the cardiomyocytic differentiation of ES cells in vitro and that Cx40 expression may be linked to early stages in cardiomyocytic differentiation.