P19 embryonal carcinoma stem cells have been used as a model system in studies on the molecular mechanisms underlying cardiomyogenic differentiation. To date, however, the efficacy of P19 cells to differentiate into cardiomyogenic lineage still low. We examined the effects of different culture conditions such as different concentrations of serum and 5-azacytidine, and culture period on cardiomyogenic differentiation of P19 cells to develop an efficient protocol for directing the cardiomyogenic differentiation of P19 cells. In some experiments, cardiomyogenic differentiation was also monitored in P19 cells transfected with cardiac-specific myosin light chain (MLC) promoter-driven DsRed reporter gene. P19 cells were plated at a density of 1 �� 10⁶ cells on 10-cm bacterial dishes for 96 h in the presence of 1% DMSO to form embryoid bodies which generate cardiac lineage cells. The formed embryoid bodies were transferred into 24-well plates, and cultured in DMEM with 2% or 10% FBS for further period of 10 or 15 days in the presence of 0, 1 and 3 關M 5-azacytidine. Quantitative real-time PCR analysis showed that the expression of cardiac muscle-specific genes such as GATA4, MEF2C, 慣-actin, 慣-MHC and cTnT was significantly higher in the 15-day culture groups than in the 10-day culture groups. Cardiac muscle-specific genes were significantly expressed in the high serum groups than in the low serum groups. Moreover, the effect of serum concentration was more distinct in the 10-day culture groups than in the 15-day culture groups. 1 關M of 5-azacytidine treatment significantly induced the expression of cardiac muscle-specific genes, which was more remarkable in the low serum groups. Furthermore, cardiomyogenic differentiation of P19 cells was also confirmed by both the expression of DsRed reporter gene driven by cardiac-specific MLC promoter and the immunostaining with cardiac muscle-specific antibodies. Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells could be enhanced by the combination of different experimental factors as well as by the application of cardiac specific promoter-based monitoring system.