RalGDS is a guanine nucleotide dissociation stimulator for Ral, and one of its homologues is RGL (RalGDS-like). In this study, the effects of mutations of Ras and the Ras-binding domains (RBDs) of RalGDS and RGL on their binding have been systematically examined. The D33A mutation of Ras reduces the abilities to bind RGL-RBD and RalGDS-RBD. To identify the RGL residue interacting with Asp33 of Ras, double-mutant analyses between Ras and RGL-RBD were conducted. For example, the K685A mutation of RGL-RBD has a much smaller effect on the RGL-RBD binding ability of the D33A mutant than on those of other mutants of Ras. Accordingly, it is indicated that the attractive interaction of Asp33 in Ras with Lys685 in RGL-RBD (Lys816 in RalGDS-RBD) contributes to the Ras.RBD association. This interaction is consistent with the crystal structure of the complex of RalGDS-RBD and the E31K Ras mutant [Huang, L., Hofer, F., Martin, G. S., and Kim, S.-H. (1998) Nat. Struct. Biol. 5, 422-426]. This crystal structure exhibits interactions of the mutation-derived Lys31 side chain with three RalGDS residues. Glu31 of Ras discriminates Ras from a Ras-homologue, Rap1, with Lys31, with respect to RalGDS and RGL binding; the E31K mutation of Ras potentiates the abilities to bind RGL-RBD and RalGDS-RBD. To examine the role of Glu31 of the wild-type Ras in the interaction with RGL and RalGDS, double-mutant analyses were conducted. The Ras binding ability of the E689A mutant of RGL-RBD is much stronger than that of the wild-type RGL-RBD, and the E31K mutation of Ras no longer potentiates the Ras binding ability of the E689A mutant. Therefore, the repulsive interaction between Glu31 in Ras and Glu689 in RGL-RBD (Asp820 in RalGDS-RBD) may keep the Ras.RBD association weaker than the Rap1.RBD association, which might be relevant to the regulation of the signaling network.