We have established a CGR8 embryonic stem (ES) cell clone (MLC2ECFP) which expresses the enhanced cyan variant of Aequorea victoria green fluorescent protein (ECFP) under the transcriptional control of the ventricular myosin light chain 2 (MLC2v) promoter. Using epifluorescence imaging of vital embryoid bodies (EB) and reverse transcription-polymerase chain reaction (RT-PCR), we found that the MLC2v promoter is switched on as early as day 7 and is accompanied by formation of cell clusters featuring a bright ECFP blue fluorescence. The fluorescent areas within the EBs were all beating on day 8. MLC2ECFP ES cells showed the same time course of cardiac differentiation as mock ES cells as assessed by RT-PCR of genes encoding cardiac-specific transcription factors and contractile proteins. The MLC2v promoter conferred ventricular specificity to ECFP expression within the EB as revealed by MLC2v co-staining of ECFP fluorescent cells. MLC2ECFP-derived cardiac cells still undergo cell division on day 12 after isolation from EBs but withdraw from the cell cycle on day 16. This ES cell clone provides a powerful cell model to study the signalling roads of factors regulating cardiac cell proliferation and terminal differentiation with a view to using them for experimental cell therapy.