AbstractThe tubular heart differentiates from the bilateral cardiac fields in the splanchnic mesoderm. The expression of smooth muscle proteins has been shown to accompany the early phases of cardiac muscle formation. In this study we show that during elongation of the arterial pole of the mouse linear heart tube, α‐smooth muscle actin (α‐Sma) expression extends in the area that has been shown to become recruited into the myocardial lineage, but does not yet express myocardial markers. These data suggest that α‐Sma identifies mesodermal cells that during subsequent development will be recruited into the myocardial lineage. Myocardium formation is not only observed at the arterial pole, but also at the venous pole and in the intracardiac mesenchyme. This results in the formation of the caval and pulmonary myocardium, the smooth‐walled atrial myocardium, the myocardial atrioventricular septum, and the myocardial outlet septum. To determine whether recruitment into the myocardial lineage also takes place in these regions, the spatiotemporal pattern of expression of α‐Sma and of the myocardial markers sarcoplasmatic reticulum calcium ATPase (Serca2a), α‐myosin heavy chain (Mhc), and β‐Mhc were examined. We show that prior to the expression of myocardial markers, α‐Sma is expressed in these regions, which suggests that these mesodermal cells become recruited into the cardiac lineage after formation of the linear heart tube. Anat Rec Part A 271A:303–314, 2003. © 2003 Wiley‐Liss, Inc.