Pluripotent P19 embryonal carcinoma (EC) cells have been used as a model system for studying the role of cardiac-specific transcription factors and upstream signaling pathways in cardiac cell differentiation because of their capacity to form cardiomyocytes. The atrial natriuretic factor (ANF) gene promoter, a known downstream target for the GATA-4 and Nkx2-5, is one of early markers of cardiac differentiation. In addition, a 230-bp proximal promoter region of the cardiac troponin 1 (cTn1) promoter was shown to be sufficient to drive cardiac-specific _expression in vivo and in vitro. This study is to establish stable P19 EC cell lines which express GFP molecules under the transcriptional control of the ANF or the cTn1 promoter in order to monitor cardiac differentiation in vitro and enrich cardiomyocytes by fluorescence-activated cell sorting (FACS). A 785-bp fragment of ANF and a 408-bp fragment of cTnI promoter were amplified using the ge! nomic DNA isolated from ICR mice and cloned into pEGFP-1 vector, respectively. The clones were transfected into undifferentiated P19 EC cells and selected for 3 weeks with G418. Ten neomycin-resistant colonies of P19 EC cells per each construct were further selected. To ensure that EGFP was stably expressed under the control of cardiac-specific promoters, ANF-EGFP or cTn1-EGFP P19 EC cells were allowed to differentiate for 5 days within embryonic bodies (EBs). Several clones showing the brightest GFP _expression were finally selected after 9 days of differentiation. Immunocytochemistry showed that P19 EC cells expressing GFP also co-expressed the cardiac specific proteins such as the cardiac myosin heavy chain and sarcomeric 慣-actinin. These P19 EC cell lines could be efficiently used as a model system to study molecular mechanisms that control the cardiac cell differentiation in vitro.