Aberrant regulation of smooth muscle cell proliferation and migration is associated with the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth muscle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thrombin peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [3H]thymidine incorporation. Both ligands also increased cell migration as measured by the Boyden chamber method. L-Phenylephrine failed to induce either of these responses but increased inositol phosphate accumulation and mitogen-activated protein kinase activation in these cells, which indicated that the cells were responsive to alpha1-adrenergic stimulation. The C3 exoenzyme, which ADP-ribosylates and inactivates Rho, fully inhibited both thrombin-stimulated proliferation and migration but had no effect on inositol phosphate accumulation. In addition, Y-27632, an inhibitor of the Rho effector p160ROCK/Rho kinase, decreased thrombin-stimulated DNA synthesis and migration. To directly examine Rho activation, Rho-[35S]GTPgammaS binding was measured. The addition of the thrombin peptide SFLLRNP, but not phenylephrine, to RASMC lysates resulted in a significant increase in Rho-[35S]GTPgammaS binding. Thrombin and SFLLRNP, but not phenylephrine, also increased membrane-associated Rho in intact RASMCs, consistent with selective activation of Rho by thrombin. These results indicate that thrombin activates Rho in RASMCs and establish Rho as a critical mediator of thrombin receptor effects on DNA synthesis and cell migration in these cells.