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The P19 embryonal carcinoma cell line is a use-ful model cels for studies on cardiac difer-entiation. However, its low eficacy of diferen-tiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cels to differentiate into a high-efficacy cardiomyo-cytes. Embryoid-body-like structures were formed after 6 days with 1 M of 5-aza in a P19 cell monolayer culture, beating cel clusters first ob-served on day 12, and, the production of beat-36-wells) after 18 days. In comparison, the spon-taneous beating cels was 3.3% (12 of 36-wels) for the untreated control cels. In response to 1 M of 5-aza, P19 cels expressed bone mor-phogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac tro-ponin I, and desmin were up-regulated in a time-dependent maner after induction of BMP signaling molecules. Imunocytochemistry re-vealed the expression of smoth muscle αsarcomeric α-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric α-actin positive cels acounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces diferentiation of P19 cels into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sul-foxide exposure, depending in part on alteration gest that 5-aza treatment could be used as a new method for cardiac diferentiation in P19 cells.