Three α-muscle actin isoforms are sequentially expressed during in vivo cardiac development. α-Smooth muscle actin is first and transiently expressed, followed by α-skeletal and finally α-cardiac actin. The significance of these transitions in actin gene expression during myogenesis remains to be determined. To understand whether actin isoforms have specific functions during cardiac development and cardiomyocyte contractility, we have hampered α-smooth muscle and α-skeletal actin expression and organization during embryonic stem cell differentiation towards cardiomyocyte. We show that the sequence of actin isoform expression displays similar pattern in the in vitro model and in mouse heart embryogenesis. Treatment with an interfering fusion peptide containing the N-terminal sequence of α-smooth muscle actin during a time window preceding spontaneous beating, prevents proper cardiac sarcomyogenesis, whereas α-skeletal actin-fusion peptide has no effect. Knockdown of α-smooth muscle actin in embryonic stem cells using RNA interference also affects cardiac differentiation. The application of both fusion peptides on beating embryoid bodies impairs frequency. These results suggest specific functional activities for actin isoforms in cardiogenesis and cardiomyocyte contractility.