Objective: To determine the role of rho signaling in sphingosine-1-phosphate (S-1-P)-induced smooth muscle cell migration. Background: S-1-P, a bioactive sphingolipid released from activated platelets, stimulates migration of smooth muscle cells (SMCs) in vitro through Gαi G-proteins and MAPK activation. Rho is one of the key small GTPases required for cytoskeletal reorganization and MAPK activation during migration. We hypothesized that S-1-P-stimulated migration is regulated by the rho signaling pathway. Methods: Rat arterial SMCs were cultured in vitro. Linear wound assays of migration were performed in the presence of S-1-P with and without C3 (a rho antagonist) or Y27632 (a rho kinase inhibitor). Western blotting was performed for ERK1/2 and p38MAPK phosphorylation after stimulation with S-1-P, with and without pre-incubation with these inhibitors. Statistics were analyzed by one-way ANOVA. Results: S-1-P stimulated migration of SMCs in a wound assay (two-fold over control; P < 0.01), which was blocked by rho inhibition (P < 0.05) but enhanced by rho kinase inhibition (P < 0.05). S-1-P induced time-dependent increases in ERK1/2 and p38MAPK phosphorylation. In the presence of C3, ERK1/2 phosphorylation was significantly decreased, while p38MAPK phosphorylation was unchanged. In contrast, rho kinase inhibition by Y27632 resulted in an increase in ERK1/2 phosphorylation and a decrease in p38MAPK phosphorylation. Conclusions: S-1-P differentially regulates MAPK pathways through components of the rho pathway. Rho regulates ERK1/2 activation, while rho kinase negatively modulates ERK1/2 and positively regulates p38MAPK. This is the first description of differential MAPK regulation by a G-protein coupled receptor through the rho pathway. Understanding signal transduction in smooth muscle cells will contribute to the development of molecular therapeutics for intimal hyperplasia.