The mammalian heart develops from a primary heart tube, which is formed by fusion of bilateral cardiac territories in which myocardial and endothelial cells have already begun to differentiate from splanchnic mesoderm. A population of myocardial precursors has been identified in pharyngeal mesoderm, anterior to the early heart tube. Cell labeling studies have indicated that this novel territory, called the anterior heart field (AHF), gives rise to the myocardial wall of the outflow tract. We now report that not only the myocardium of the outflow tract but also myocardial cells of the embryonic right ventricle are derived from this source. Explants of pharyngeal mesoderm or of the early heart tube were cultured from transgenic mice in which transgene expression marks different regions of the heart. Pharyngeal mesoderm from 5 to 7 somite embryos gives rise to cardiomyocytes with right ventricular and outflow tract identities, whereas the heart tube as this stage has an essentially left ventricular identity. DiI labeling confirms that the early heart tube is destined to contribute to the embryonic left ventricle and indicates that right ventricular myocardium is added from extracardiac mesoderm. Retrospective clonal analysis of the heart at embryonic day (E) 10.5 reveals the existence of a clonal boundary in the interventricular region, which appears during ventricular septation, underlining different origins of the two ventricular compartments. This study demonstrates the differences in the embryological origin of right and left ventricular myocardium, which has important implications for congenital heart disease.