After the development of the linear heart tube, additional myocardium is formed leading to the muscular mantle around the caval and pulmonary veins and the muscular septa in the embryonic heart. Here, we report the results of our in vivo and in vitro studies of this late myocardium-generating process in the mouse. By using an immunohistochemical approach, we determined that myocardium formation starts around embryonic day 12 in the dorsal mesocardium. In subsequent stages of development, the process extends downstream into the intracardiac mesenchymal tissues of the atrioventricular canal and outflow tract and upstream into the extracardiac mediastinal mesenchyme embedding the pulmonary and caval veins. Given the spatiotemporal pattern of myocardium formation, we applied a three-dimensional in vitro explant culture assay to investigate the myocardium-generating potential of the different cardiac compartments. We determined that this potential is stage- and mesenchyme-dependent. This latter finding suggests an important role for mesenchyme in myocardium formation after the development of the linear heart tube. Developmental Dynamics 227:1–13, 2003 © 2003 Wiley-Liss, Inc.