RalA and RalB are GTPases of unknown function and are activated by proteins, RalGDS, that interact with the active form of another GTPase, Ras. To elucidate Ral function, we have searched for proteins interacting with an activated form of RalA using the two-hybrid method and a Jurkat cell library. We have identified a partial cDNA encoding a protein, RLIP1, which binds to activated RalA and this binding requires an intact effector domain of RalA. Biochemical data with purified RalA confirm the genetic results. This protein also bears a region of homology with GTPase-activating protein (GAP) domains that are involved in the regulation of GTPases of the Rho family and, indeed, RLIP1 displays a GAP activity acting upon Rac1 and CDC42, but not RhoA. This GAP region is not required for RLIP1 binding to Ral.The whole cDNA was cloned, and it encodes a 76-kDa polypeptide, RLIP76, which also binds RalA. The Rho pathway is involved in membrane and cytoskeleton modifications after mitogenic stimulation and acts in parallel to and synergistically with the Ras pathway. We propose that these pathways are linked through a cascade composed of Ras 3 RalGDS 3 Ral 3 RLIP76 3 CDC42/Rac1/Rho, allowing modulation of the Rho pathway by the Ras pathway.