RalGPS2 is a murine guanine nucleotide exchange factor of the RalGPS family; it contains a Cdc25-like GEF domain and does not exhibit a Ras-binding domain. The main characteristic of RalGPS2 is its pleckstrin homology (PH) domain, present at the C terminus, that preferentially binds phosphatidylinositol-4,5-biphosphate and in HEK 293 cells localized in membranes, causing ruffling and vesiculation. Moreover, RalGPS2 contains a PxxP motif in the central part of the molecule. This motif binds in vitro and in vivo SH3 domains of Grb2 and PLCgamma. RalGPS2 and its GEF domain activate RalA in vivo while the PH-PxxP domains inhibited it behaving as a dominant negative for the RalA pathway; this activation was not inhibited by co-expression of a dominant negative Ras. RalGPS2 is physiologically expressed in testis and brain; when overexpressed, the whole RalGPS2 causes considerable morphological changes in HEK 293 cells, suggesting its possible role on cytoskeleton reorganization. This is further strengthened by data obtained in NIH3T3 cells where expression of PH-PxxP domain promotes actin depolymerization. Finally, RalGPS2 and its GEF domain induce Ras-independent transcriptional activation of the c-fos promoter in NIH3T3 cells.