AbstractTo aid in detection and tracking of cells targeted by the left‐right (LR) pathway in the heart throughout morphogenesis, expression from a Pitx2c‐lacZ transgene (P2Ztg) was analysed in detail. β‐galactosidase expression from P2Ztg was robust, allowing reliable visualisation of low‐level Pitx2c expression, and was virtually entirely dependent upon NODAL signalling in the heart. P2Ztg showed expression in trabecular and septal, as well as non‐trabecular, myocardium, and a strong expression bias in myocardium associated with individual endocardial cushions of the atrioventricular canal and outflow tract, which are essential for cardiac septation. Expression on the ventral surface of the outflow tract evolved to a specific stripe that could be used to track the future aorta during outflow tract spiralling and remodelling. Our data show that the P2Ztg transgene is a useful resource for detection of molecular disturbances in the LR cascade, as well as morphogenetic defects associated with other cardiac congenital disorders. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.