alpha v beta 3 integrins have been implicated in regulating vascular healing in animal models of arterial injury. Because the specific cellular events mediated by alpha v beta 3 integrins are not completely understood, we examined alpha v beta 3 integrin-dependent cytoplasmic events in cultured human smooth muscle cells (SMC) following treatment with thrombospondin-1 (TSP), a glycoprotein concentrated at sites of blood vessel injury. TSP treatment elicited a time-dependent association of nonmuscle myosin heavy chain-A (NMHC-A) with alpha v beta 3 integrins. NMHC-A also associated with focal adhesion kinase (FAK) in TSP-treated SMC. FAK, a nonreceptor kinase implicated in integrin-mediated signaling, was phosphorylated on tyrosine in growth-arrested SMC, but levels of tyrosine phosphorylation increased following treatment with TSP. To test whether NMHC-A was regulated by vascular injury, we examined expression in baboon brachial arteries. In uninjured arteries, NMHC-A staining was present in the media. In arteries injured by balloon withdrawal, medial NMHC-A expression was increased with intense staining at specific sites. In summary, heteromeric protein complexes involving alpha v beta 3 integrins, NMHC-A, and FAK form following treatment of human SMC with TSP. These results suggest that the formation of protein signaling complexes is one mechanism whereby alpha v beta 3 integrins influence intracellular signaling pathways.