Previous studies have shown that the cells of the aorticopulmonary (AP) septum are similar to the smooth muscle cells of the mediae of the great vessels in their common origin from the cardiac neural crest and in their common expression of an elastic extracellular matrix. The purpose of this study was to test the cells of the AP septum for the presence of certain cytoplasmic proteins, especially smooth muscle alpha-actin (SMAA) whose presence is definitive of smooth muscle. A monoclonal antibody against SMAA was applied to normal chicken embryos at 3.5-8 days of incubation and to age-matched embryos from which the cardiac neural crest had been ablated surgically. Antibodies against the intermediate filaments desmin, cytokeratin, and vimentin also were applied. The results showed that the AP septal cells expressed SMAA during the process of septation, days 5-8; but when the cardiac neural crest was ablated and septation was defective, no cells in the conotruncal connective tissue expressed SMAA. None of the intermediate filament proteins were detected in the septum. These results indicate that the AP septal cells are smooth muscle and therefore may be hypothesized to have an active role in septation.