The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (ko(myo)) we show it has a crucial role in this process. Pitx2 ko(myo) mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 ko(myo) hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 ko(myo) mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.