Background: Recent studies have reported the existence of resident cardiac stem cells (CSCs) that have potential differentiating into cardiomyogenic and endothelial cell lineage, and therefore promising candidates for cell therapy in cardiovascular field. However, a drawback is their limited life span during in vitro culture. Therefore, we constructed the retroviral vector expressing human telomerase reverse transcriptase (hTERT)-IRES eGFP to establish immortalized CSC lines. Methods and Results: Retroviruses were produced in 293GPG packaging cells by transfection with retroviral vectors carrying the hTERT-IRES eGFP genes. CSCs were isolated by collagenase treatment from heart of ICR mice and infected with retroviruses harboring the hTERT-IRES eGFP genes. The putative CSC lines were selected in the medium containing puromycin for about 3 months, and sorted on a fluorescence-activated cell sorter by strong eGFP expression. The three hTERT-immortalized CSC lines derived from a single cell were finally selected in 96-well cell culture plates. They showed strong eGFP expression and high levels of hTERT activity as well as retained as normal diploid karyotypes after an extended culture period more than 4 months. The two CSC lines expressed the mesenchymal stem cell markers CD29, CD44, CD71, CD106 and Sca-1 but not CD14, CD31, CD34, CD45, CD90, CD117 and CD133. Interestingly, the one CSC line strongly expressed the endothelial cell marker CD31 with similar expression patterns of the mesenchymal stem cell markers. The all three CSC lines retained multi-potential differentiating into cardiomyocytes, endothelial cells, adipocytes and osteocytes but the CD31⁺ CSC cell line especially showed high endothelial potential than those of the other two CSC lines. Conclusions: This result shows that the tissue-specific cell types with different phenotypic characteristics have different potential for specific cell lineages. The hTERT-immortalized cardiac stem cell lines are very useful for the study of stem cell differentiation as well as for cell and gene therapy in cardiovascular field.