Ral, a member of small GTP-binding protein (G protein) superfamily, has been suggested to act downstream of Ras, since Ral GDP dissociation stimulator (RalGDS) has been found to be an effector protein of Ras. In this study, we examined the effects of RalGDS and Ral on gene expression using c-fos promoter linked to the luciferase reporter gene (c-fos-luciferase). RalGDS interacted with RasG12V/E37G (in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf in COS cells. RafCAAX is an active Raf kinase targeted to the plasma membranes by virtue of the addition of a C-terminal localization signal from K-Ras. Transfection of either RalGDS or RafCAAX into NIH3T3 cells slightly stimulated c-fos-luciferase expression and cotransfection of both proteins greatly enhanced the expression. RalGDS and an activated Rac (RacG12V) did not act synergistically to stimulate c-fos-luciferase expression. Transfection of an activated Ral (RalG23V) stimulated c-fos-luciferase expression. Furthermore, cotransfection of RalG23V and an activated Ras (RasG12V) enhanced RasG12V-dependent c-fos-luciferase expression. However, RalG23V did not synergize with RafCAAX, RacG12V or RalGDS to stimulate the expression. These results show that RalGDS and Ral regulate c-fos promoter activity and suggest that RalGDS may activate c-fos promoter synergistically with the signal from Raf by transmitting the signal to a target other than Ral.