p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of p160ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).