Ral-binding protein 1 (RalBP1) is a putative effector protein of Pal and possesses the GTPase-activating activity for Rac1 and CDC42. We examined the roles of the posttranslational modifications of Ral and Rac1 for the action of RalBP1. In COS cells, Ral(G23V), a constitutively active form, was mainly detected in the membrane fraction while most of Ral(G23V/C203S), a Ral mutant which is not post-translationally modified, was found in the cytosol fraction, When RalBP1 was expressed alone in COS cells, it was found in the cytosol but not in the membrane fraction, When RalBP1 was coexpressed with Ral(G23V), a part of RalBP1 was found in the membrane fraction, However, when RalBP1 was coexpressed with Ral(G23V/C203S), all of RalBP1 was recovered in the cytosol fraction, Although Ral bound to RalBP1 at a molar ratio of 1:1, the interaction of Ral with RalBP1 did not affect the GTPase-activating activity of RalBP1 for Rad. Furthermore, RalBP1 was more active on the post-translationally modified form of Fact and CDC42 than the unmodified form. These results suggest that the post-translational modification of Ral is important for the subcellular localization of RalBP1 and that the interaction of Ral with RalBP1 is not essential for the activity of RalBP1 but plays a role in recruiting RalBP1 to the membrane where its substrates, Rad and CDC42, reside. (C) 1997 Federation of European Biochemical Societies.