Backgrond: Satellite cells that originate from skeletal muscle are one of the most promising candidates of cellular myoplasty. We evaluated optimal preparation and differentiation condition of satellite cells in vitro, and assessed the integration of satellite cell with cardiomyocytes in vivo condition. Subjects and methods: We compared two primary culture method of satellite cells of neonatal and adult Fisher 344 rat. Proliferation and differentiation media were introduced to induce myotube formation. Additionally, influence of Ca2+ concentration on differentiation and proliferation of satellite cells were evaluated. We analyzed differentiation pattern with RT-PCR and immunocytochemistry of pan-cadherin, myo D, Troponin-I, T and Connexin 43. As in vivo experiment, 1.5��10⁶ Di I and DAPI double labeled satellite cells were transplanted into normal rat myocardium and harvested at 2,5,10 days after injection. Results: Satellite cells obtained from single fiber method showed higher purity than mincing method(proportion of satellite cells confirmed with immunocytochemistry of myosin heavy chain: 92.5짹2.5% vs. 86.4짹2.7%, P<0.05). On RT-PCR analysis expression of connexin 43 and pan-cadherin that are constitutively expressed on satellite cells, declined as differentiation progressed. Expression of myo D was confined in late proliferation and early differentiation phase. Expression of cardiac Troponin was not observed. Ca2+ concentration did not affect outcomes of primary culture and differentiation induction. Proportion of beating myotube was higher in culture condition with serum concentration above 10% and Ca2+ concentration of 0.18mM. After transplantation, by 5 days, the graft cells integrated and aligned with native cardiomyocytes. At 10 days, immunohistochemistry demonstrated that satellite cells expressed connexin 43 between satellite cells and native cardiomyocytes. Conclusion: Single fiber method is a feasible and efficient method of preparation of satellite cells. Satellite cells from adult rats were comparable with cells from neonatal rats in proliferation and differentiation. Satellite cell is an eligible candidate of cellular myoplasty.