R-Ras has a high degree of sequence homology with Ras and other members of the Ras subfamily, including Rap, TC21, and M-Ras. Although R-Ras has been suggested to regulate cell adhesion, migration, and invasion, the biological mechanism has not been well assessed. In this report, we show that constitutively active R-Ras (38V) induces a more rounded cell shape and redistribution of focal adhesion, and enhances the phosphorylation of focal adhesion kinase and paxillin. Active R-Ras (38V) induces cell adhesion to type I collagen, but inhibits cell motility. In active R-Ras (38V) cells, the activity of RhoA is increased and accompanied with translocation to plasma membrane, but not that of Rac1 or Cdc42. In parallel, dominant-negative RhoA (N19RhoA) and Y27632, a specific inhibitor of Rho-associated kinase, dramatically reverse the rounded cell morphology to a spread cell shape and enhance motility. Furthermore, coincident with the formation of cortical actin filaments in active R-Ras (38V) cells, myosin light chain and Ser-19-phosphorylated myosin light chain mainly accumulate at the peripheral region, which is inhibited by the treatment of Y27632. Using H-Ras/R-Ras and R-Ras/H-Ras hybrid constructs, we show that the COOH-terminal region of R-Ras contains the specific signal for inducing changes in motility and morphology. Our results suggest that R-Ras in breast epithelial cells disrupts cell polarity and motility through the Rho/Rho-associated kinase pathway triggered by a signal from the COOH-terminal end of R-Ras.