Differentiation cultures of embryonic stem (ES) cells can be a useful in vitro system for understanding cardiac myocyte development. However, cell morphometry, sarcomere development, and functional cell-cell junction formation have not been examined in detail to determine whether ES cell-derived cardiac myocytes exhibit structural and functional characteristics similar to cardiac myocytes within the developing heart. Therefore, we examined cellular dimensions, sarcomere formation, and cell-cell contacts in differentiating cardiac myocytes derived from mouse D3-ES cell cultures. Cells exhibited rod-shaped morphology and had single centrally located nuclei, typical of maturing cardiac myocytes. The cellular dimensions of 59 individual cardiac myocytes within contracting foci of ES cell cultures were analyzed (length 5 42.2 6 2.1 Β΅m, area 5 197 6 19 Β΅m 2 , and diameter 5 5.5 6 0.3 Β΅m) and found to be similar to myocytes in vivo. Transmission electron micrographs of ES cell-derived cardiac myocytes indicated myofibrillar architecture ranged from sparse and disorganized to densely packed, parallel arrays of myofibrils organized into mature sarcomeres. This pattern of myofibrillar assembly in maturing sarcomeres was similar to that observed during in vivo myocyte differentiation. Another hallmark of cardiac development is the formation of intercalated discs, which functionally couple adjacent cardiac myocytes. Electron micrographs indicated nascent intercalated discs were forming in foci of ES cell-derived cardiac myocytes. In addition, indirect immunostaining with anti-connexin 43 antibody (Ab), a monoclonal Ab to the gap junction component of the intercalated disc, indicated that gap junctions were present in contracting ES cell foci. Furthermore, microinjection of single cardiac myocytes with Lucifer yellow (2.5 Β΅M) resulted in the spread of fluorescence to adjacent cells within a contracting focus, an indication of functional cell-cell coupling across these gap junctions. Together, these results indicate ES cell-derived cardiac myocytes exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo. Cell Motil.