RalGDS family members (ralGDS and RGL) interact with the GTP-bound form of Ras through its effector loop. The C-terminal region (amino acids 602-768) of RGL is responsible for binding to Ras. In this paper we characterized a Ras-interacting domain of RGL using deletion mutants of RGL(602-768). RGL(602-768), RGL(632-768), and RGL (602-734) bound to the GTP-bound form of Ras and inhibited the GAP activity of NF-1. RGL(646-768) showed a low binding activity to Ras and inhibited GAP activity of NF-1 weakly. None of RGL(659-768), RGL(685-768), RGL(602-709), and RGL(602-686) bound to Ras or inhibited GAP activity of NF-1. These results indicate that amino acids 632-734 of RGL constitute a nearly minimal domain that contains the binding element for Ras. RGL(632-734) inhibited v-Ras- but not progesterone-induced Xenopus oocyte maturation. Furthermore, RGL(632-734) inhibited v-Ras- but not v-Raf- dependent extracellular signal-regulated kinase activation in Xenopus oocytes. These results clearly demonstrate that the Ras-interacting domain of RGL is important for Ras-dependent signal transduction in vivo.