This study describes a method to generate pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene, consisting of the α-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase, was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytochemical and ultrastructural analyses confirmed that the selected cardiomyocyte cultures (>99% pure) were highly differentiated. G418-selected cardiomyocytes were tested for their ability to form stable intracardiac grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistochemistry and PCR analysis, which revealed the presence of ES-derived cardiomyocyte grafts for up to 7 weeks after implantation. This study demonstrates that a simple genetic manipulation can be used to generate pure cultures of cardiomyocytes from differentiating ES cells, which are suitable for cardiac engraftment. The selection approach is applicable to all ES-derived cell lineages.This study describes a method to generate pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene, consisting of the α-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase, was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytochemical and ultrastructural analyses confirmed that the selected cardiomyocyte cultures (>99% pure) were highly differentiated. G418-selected cardiomyocytes were tested for their ability to form stable intracardiac grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistochemistry and PCR analysis, which revealed the presence of ES-derived cardiomyocyte grafts for up to 7 weeks after implantation. This study demonstrates that a simple genetic manipulation can be used to generate pure cultures of cardiomyocytes from differentiating ES cells, which are suitable for cardiac engraftment. The selection approach is applicable to all ES-derived cell lineages.