Arterial smooth muscle cells produce large amounts of extracellular matrix molecules during repair processes and in primary culture. This occurs after a transition of the cells from a contractile to a synthetic phenotype and together with the acquisition of proliferative capacity. Here, the deposition of the glycoprotein tenascin in the extracellular matrix of rat arterial smooth muscle cells in vivo and in vitro was studied by immunofluorescence microscopy and immunoblotting. Tenascin was found in the intimal layer of the adult rat aorta and carotid artery, but not in the media. Tenascin also appeared in the neointima formed by proliferating smooth muscle cells 2 weeks after balloon catheter injury of the carotid artery. To determine if the deposition of tenascin in the neointima after arterial injury was related to changes in smooth muscle function, freshly isolated rat aortic smooth muscle cells were seeded on a substrate of plasma fibronectin and allowed to modulate from a contractile to a synthetic state in a serum-free medium. In these quiescent cultures, a tenascin-containing extracellular matrix was formed after 3 to 4 days. Taken together, the results show that tenascin production is induced concomitantly with changes in smooth muscle phenotype both in vivo and in vitro.