We recently isolated a cardiomyogenic (CMG) cell line from murine bone marrow stroma, and in this paper characterize regenerated cardiomyocytes derived from adult mesenchymal stem cells at the molecular level. Stromal cells were immortalized, exposed to 5-azacytidine, and repeatedly screened for spontaneously beating cells. CMG cells began to beat spontaneously after 2 weeks, and beat synchronously after 3 weeks. They exhibited sinus-node-like or ventricular-cell-like action potentials. Analysis of the isoforms of contractile protein genes, such as of myosin and alpha-actin, indicated that their phenotype was similar to that of fetal ventricular cardiomyocytes. The cells expressed Nkx2.5, GATA4, TEF-1, and MEF2-C mRNA before 5-azacytidine exposure, and MEF2-A and MEF2-D after exposure. CMG cells expressed alpha1A, alpha1B, and alpha1D-adrenergic receptor mRNA prior to differentiation, and beta1, beta2-adrenergic and M1, M2-muscarinic receptors after acquiring the cardiomyocyte phenotype. Phenylephrine induced phosphorylation of ERK1/2, and the phosphorylation was inhibited by prazosin. Isoproterenol increased the cAMP level 38-fold and beating rate, cell motion, %shortening, and contractile velocity by 48%, 38%, 27%, and 51%, respectively, and the increases were blocked by CGP20712A (beta1-selective blocker). Carbachol increased IP3 32-fold, and the increase was inhibited by AFDX116 (M2-selective blocker). These findings demonstrated that the regenerated cardiomyocytes were capable of responding to adrenergic and muscarinic stimulation. This new cell line provides a model for the study of cardiomyocyte transplantation.