Introduction: Skeletal myoblasts were extensively explored for cardiac repair. However, skeletal myoblasts had only limited evidences of functional and structural differentiation to cardiomyocytes. In this study, we evaluated differentiation potentials of skeletal myoblasts and the influence of co-culture with cardiomyocytes on trans-differentiation of skeletal myoblasts. Subjects and methods: Skeletal myoblasts from Fisher 344 rats were isolated by single fiber method and evaluated for functional differentiation to cardiomyocytes. Differentiation-induction media and co-culture with cardiomyocytes were used to induce differentiation of skeletal myoblasts. Differentiation status was evaluated by RT-PCR and immunocytochemistry of cardiomyocyte-specific genes, troponins-I, troponins-T, connexin-43, and N-Cadherin. Presence and nature of spontaneously beating cells and responses to adrenergic stimulation were evaluated simultaneously. Results: Under single culture condition of skeletal myoblasts, neither expression of cardiac specific cytoplasmic proteins was induced nor spontaneously beating single cells were observed. But, co-culture of skeletal myoblast with cardiomyocytes induced expression of troponins and produced spontaneously beating single cells originating from skeletal myoblasts. Spontaneous beating ���myotubes��� were observed in both differentiation conditions. But positive or negative chronotropic response to adrenergic agonist (isoproterenol) or antagonist (propranolol) was observed only in skeletal myoblast from co-culture condition. Conclusion: Co-culture with cardiomyocytes is essential to induce functional differentiation of skeletal myoblasts to cardiomyocytes.