Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.