AbstractThe recent discovery of several myogenic cardiac progenitor cells in the post‐natal heart suggests that some myocardial cells may remain undifferentiated during embryonic development. In this study, we examined the subcellular characteristics of the embryonic (E) mouse ventricular myocardial cells using transmission electron microscopy (TEM). At the ultrastructural level, we identified three different cell populations within the myocardial layer of the E11.5 heart. These cells were designated as undifferentiated cells (43 ± 6%), moderately differentiated cells (43 ± 2%) and mature cardiomyocytes (14 ± 4%). Undifferentiated cells contained a large nucleus and sparse cytoplasm with no myofibrillar bundles. Moderately differentiated cells contained randomly arranged myofilaments in the cytoplasm. In contrast, mature cardiomyocytes contained well‐developed sarcomere structures. We also confirmed the presence of similar undifferentiated cells albeit at low levels in the E16.5 (∼20%) and E18.5 (∼7%) myocardium. Further we used immunogold labeling technique to test whether these distinct cell populations were also positive for markers such as Nkx2.5, ISL1 and ANF. A preponderance of anti‐Nkx2.5 label was found in the undifferentiated and moderately differentiated cell types. Anti‐ANF label was found only in the cytoplasmic compartment of moderately differentiated and mature myocardial cells. All of the undifferentiated cells were negative for anti‐ANF labeling. We did not find immuno‐gold labeling with ISL1 in any of the three myocardial cell types. Based on these results, we suggest that embryonic myocardial cell differentiation is a gradual process and undifferentiated cells expressing Nkx2.5 in post‐chamber myocardium may represent a progenitor cell population while cells expressing Nkx2.5 and ANF represent differentiating myocytes.