The activity of Raf-1 and Rok-{alpha} kinases is regulated by intramolecular binding of the regulatory region to the kinase domain. Autoinhibition is relieved upon binding to the small guanosine triphosphatases Ras and Rho. Downstream of Ras, Raf-1 promotes migration and tumorigenesis by antagonizing Rok-{alpha}, but the underlying mechanism is unknown. In this study, we show that Rok-{alpha} inhibition by Raf-1 relies on an intermolecular interaction between the Rok-{alpha} kinase domain and the cysteine-rich Raf-1 regulatory domain (Raf-1reg), which is similar to Rok-{alpha}'s own autoinhibitory region. Thus, Raf-1 mediates Rok-{alpha} inhibition in trans, which is a new concept in kinase regulation. This mechanism is physiologically relevant because Raf-1reg is sufficient to rescue all Rok-{alpha}¿dependent defects of Raf-1¿deficient cells. Downstream of Ras and Rho, the Raf-1¿Rok-{alpha} interaction represents a novel paradigm of pathway cross talk that contributes to tumorigenesis and cell motility.,