We previously reported the cloning of a serine/threonine kinase, PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z-s., and Lim, L. (1994) Nature 367, 40-46). These p21 proteins together with RhoA comprise the Rho subfamily of proteins that are involved in morphological events. We now report the isolation of a rat cDNA encoding a 150-kDa protein, which specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROK alpha (for ROK alpha, RhoA-binding kinase), recognized a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibited serine/threonine kinase activity. In cultured cells, immunoreactive p150 was recruited to membranes upon transfection with dominant positive RhoAV14 mutant and was localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROK alpha as an effector for RhoA.