alpha-Smooth-muscle actin (SMA) is the major isoform of adult vascular tissues. During early development, SMA is expressed in various mesodermally derived tissues in a spatiotemporally restricted manner; however, its exact role remains unknown. We examined its role in the formation of chicken atrioventricular (AV) endocardial cushion tissue. This developmental process possesses the characteristics of endothelial-mesenchymal transformation and is partly TGF beta-dependent. Immunohistochemistry showed that SMA was (1) expressed homogeneously in the newly formed appendages of transforming endothelial/mesenchymal cells, and (2) distributed in a punctate manner in the lamellipodia/filopodia of invading mesenchymal cells. Antisense oligodeoxynucleotide (ODNs) specific for SMA reduced both SMA expression and mesenchymal formation in AV endothelial cells cultured with myocardium on a collagen gel lattice. Perturbation of SMA by antisense ODN also inhibited TGF beta-inducible migratory appendage formation in a cultured AV endothelial monolayer. However, it did not inhibit cell:cell separation or cellular hypertrophy. These results suggest that the expression of SMA is necessary for migratory appendage formation during the TGF beta-dependent initial phenotypic changes that occur in endothelial-mesenchymal transformation.