No consensus exists regarding the precise contribution of myocardium and the atrioventricular (AV) cushion mesenchyme to the development of leaflets, tendinous cords (TCs) and papillary muscles. Furthermore, the origin and fate of the myocardium embedded in the immature mesenchyme of the AV cushions at the beginning of AV valvulogenesis is controversial. Some authors have suggested that these cells result from a mesenchyme-to-myocardium transformation. In contrast, other researchers have concluded that they are derived from the myocardial ventricular wall and the interventricular septum (IVS). On the other hand, it has been assumed that the AV mural and septal leaflets have the same pattern of development. However the supporting structures of the two types of leaflets are anatomically different, which could reflect some differences in the pattern of development. We have therefore investigated the morphogenetic processes involved in sculpting and maturation of the right septal leaflet (RSL) and TCs in embryonic and post-hatching chicken hearts. The origin and fate of the myocardium embedded in the immature cushion mesenchyme at the beginning of RSL morphogenesis was also studied. For this purpose, scanning electron microscopic analysis, histological studies and immunohistochemical detection of Nkx2.5 and MEF2C were performed. Our findings indicate that the RSL and TCs present a distinct morphogenetic pattern from that of the mural leaflets. Our results also provide evidence that myocardial recruitment from the IVS, but not mesenchyme-to-myocardium transformation, participates in the development of the muscular region of the TCs adjacent to the IVS. Anat Rec, 2010. © 2009 Wiley-Liss, Inc.