P19 embryonal carcinoma (EC) cells have been successfully used for studies of cardiac differentiation. However, the low efficacy of smooth muscle differentiation (SMC) represents a major limitation of this system. In addition, aggregation is required for DMSO-induced differentiation of P19 EC cells into cardiac myocytes. It has been reported that cardiomyocytes can be generated from marrow stromal cells after exposure to 5-azacytidine (5-aza), a cytosine analog. We have investigated whether 5-aza can induce differentiation of P19 EC cells into cardiomyocytes in the absence of prior embryoid body (EB) formation. Undifferentiated P19 EC cells were directly exposed to different concentrations (1, 5 and 10 uM) of 5-aza without aggregate formation. The growth properties and cardiac differentiation of P19 EC cells among groups treated with different concentrations of 5-aza were analyzed by cell counting, RT-PCR and immunocytochemistry. RT-PCR analysis showed that P19 EC cells treated with 1 uM 5-aza for 10 days in vitro expressed markers characteristics of cardiomyocytes, such as cardiac 棺-myosin heavy chain, atrial natriuretic factor (ANF), desmin, and cardiac transcription factors GATA-4, Nkx2.5. However, more than 90% of P19 EC cells incubated with 5-aza at 5 uM and 10 uM necrosed. Sarcomeric 慣-actinin, desmin and cardiac myosin heavy chain were detected in 5-aza treated P19 EC cells by immunocytochemistry, confirming that 5-aza can induce differentiation of P19 EC cells to cardiomyocytes. Furthermore, the P19-derived cardiomyocytes can be dissociated and enriched by Percoll density centrifugation to give pure populations. This result suggests that the generation method of cardiomyocytes from P19 EC cells by 5-aza treatment provides a powerful model for the study of cardiomyocyte differentiation.