Although Rho, a small GTPase, has been demonstrated to play an important role in the smooth muscle contraction and relaxation, little is known about the involvement of Rho protein in smooth muscle cell (SMC) migration. In this study the role of Rho-Rho kinase pathway was examined in SMC migration induced by platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA). C3 transferase, a specific inhibitor of Rho, blocked SMC migration induced by PDGF and LPA. Y-27632, a specific inhibitor of Rho kinase, a direct target molecule of Rho, inhibited PDGF and LPA-induced SMC migration in a concentration dependent manner. Although rapid increase in myosin light chain (MLC) phosphorylation in SMC treated with LPA was observed, no enhanced MLC phosphorylation was detected in response to PDGF. Y-27632 suppressed LPA-induced as well as basal level of MLC phosphorylation. ML-9, a specific inhibitor of myosin light chain kinase (MLCK), inhibited PDGF and LPA-induced SMC migration without the suppression of MLC phosphorylation at 5 min incubation, suggesting that MLCK may contribute to SMC migration via mechanism other than MLC phosphorylation. These results suggest that Rho-Rho kinase pathway is implicated in SMC migration and that different signaling pathways downstream of Rho-Rho kinase may be involved in LPA and PDGF-induced SMC migration. MLC phosphorylation via Rho-Rho kinase pathway appears to be implicated in LPA-dependent SMC migration. Whereas PDGF-mediated SMC migration is independent of increased MLC phosphorylation and other target molecules downstream of Rho-Rho kinase seem to be involved.