Rho GTPases regulate the morphology of cells stimulated by extracellular ligands. Their activation is controlled by guanine exchange factors (GEF) that catalyze their binding to GTP. The multidomain Trio protein represents an emerging class of &Rgr; regulators that contain two GEF domains of distinct specificities. We report here the characterization of Rho signaling pathways activated by the N-terminal GEF domain of Trio (TrioD1). In fibroblasts, TrioD1 triggers the formation of particular cell structures, similar to those elicited by RhoG, a GTPase known to activate both Rac1 and Cdc42Hs. In addition, the activity of TrioD1 requires the microtubule network and relocalizes RhoG at the active sites of the plasma membrane. Using a classical in vitro exchange assay, TrioD1 displays a higher GEF activity on RhoG than on Rac1. In fibroblasts, expression of dominant negative RhoG mutants totally abolished TrioD1 signaling, whereas dominant negative Rac1 and Cdc42Hs only led to partial and complementary inhibitions. Finally, expression of a Rho Binding Domain that specifically binds RhoG(GTP) led to the complete abolition of TrioD1 signaling, which strongly supports Rac1 not being activated by TrioD1 in vivo. These data demonstrate that Trio controls a signaling cascade that activates RhoG, which in turn activates Rac1 and Cdc42Hs.