Background: Cardiac Sca-1+ and c-kit+ cells are known as cardiac stem cells or cardiac progenitors. However, characteristics and long term fate of cardiac stem cells were poorly understood. We evaluated characteristics of mouse cardiac Sca-1+ cells in vitro culture condition. Methods: Purified Sca-1+ (S1+) and – cells (S1–) were isolated from myocyte-depleted heart cells of 4 to 8 week-old C57BL/6J mice by MACS. After 5 weeks of initial culture in EGM2-MV, Sca-1+ cells were separated by MACS into Sca-1+ (S2+) and – (S2–) cells and cultured in the same condition. RT-PCR, Sca-1 FACS, proliferation assay with CFSE, and cell cycle analysis were done to evaluate Sca-1+ and – cells. Results: S1+ group expressed Nkx-2.5, GATA-4, Flk-1, VE-cadherin, calponin and 慣-smooth muscle actin, but did not express 慣-cardiac actin and c-kit. However, Flk-1 was not expressed and expression of 慣-smooth muscle actin and Nkx-2.5 were increased in S2+ and S2– group. This suggests that exposure to EGM2-MV committed freshly isolated Sca1+ cells (S1+) into myogenic stem or progenitor cells. Interestingly, S2+ and S2– groups showed similar gene expression. After 2 weeks of additional culture, FACS analysis showed S2+ and S2– groups were composed of similar ratio of Sca-1+/– cells. Cell cycle analysis and proliferation assay with CFSE also showed no difference between two cell populations. These suggest the possibility that stem cells in higher hierarchy than Sca-1+ cells, possibly ���true cardiac stem cell���, can exist. Conclusions: Mouse cardiac Sca-1+ cells showed myogenic and vasculogenic potentials and can be committed into more myogenic stem or progenitor cells with in vitro culture. The identification and characterization of ���true cardiac stem cell��� is required.