_v3 integrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Since -thrombin contributes to neointimal formation, we examined the hypothesis that _v3 integrins influence -thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed _v3 integrins as demonstrated by immunofluorescence microscopy and FACS analysis. Proliferative responses to -thrombin were partially inhibited by anti-₃ integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that -thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by _v3 antagonists. ₃ integrin staining was diffuse and did not concentrate at sites of focal adhesions. -thrombin elicited a time-dependent increase in activation of c-Jun NH2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). _v3 antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from ₃ integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not spingosphine-1-phosphate, a potent activator of Rho. In summary, _v3 integrins play an important role in -thrombin-induced proliferation and focal adhesion formation in RASMC.