Bone marrow (BM) cells are reported to contribute to the process of regeneration following myocardial infarction. The present study examined two independent clonal studies to determine the origin of bone marrow (BM)-derived cardiomyocytes. First, we transplanted single CD34(-)c-kit(+)Sca-1(+)lineage(-) side population cells (hematopoietic stem cells) from enhanced green fluorescent protein (EGFP)-transgenic mice into lethally irradiated mice, induced myocardial infarction, and treated them with G-CSF to mobilize stem cells. At 8 weeks, we could not find any EGFP(+) cardiomyocytes. In contrast, more than 5000 EGFP(+) cardiomyocytes were observed in whole BM cell-transplanted mice, suggesting that they were derived from non-hematopoietic cells. Next, clonally purified mesenchymal stem cells (MSC) that expressed EGFP in the cardiomyocyte-specific manner were transplanted directly into BM of lethally irradiated mice, and similar experiment was performed. EGFP(+) actinin(+) cells were observed in the ischemic myocardium, indicating that MSC had been mobilized and differentiated into cardiomyocytes. Together, these results suggest that the origin of the BM-derived cardiomyocytes is MSC.