We have isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneous beating cells were repeatedly screened for. The cells showed a fibroblast-like morphology. However, this morphology changed after 5-azacytidine treatment in about 30% of the cells, which connected with adjoining cells after 1 week, formed myotube-like structures and began spontaneous beating after 2 weeks, and beat synchronously after 3 weeks. These cells expressed atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Electron microscopy revealed a cardiomyocyte-like ultrastructure including typical sarcomeres and atrial granules. They had sinus node-like or ventricular cell-like action potentials. Analysis of the isoform of contractile protein genes, such as myosin and alpha-actin, indicated that their phenotype was similar to fetal ventricular cardiomyocytes. These cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine treatment, and expressed MEF2-A and MEF2-D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte transplantation.