OBJECTIVE To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application. METHODS The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/chicken beta-actin promoter/beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation. RESULTS We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the "green" ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. CONCLUSIONS The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.