Trio contains two functional guanine nucleotide exchange factors (GEF) domains for the Rho-like GTPases and a serine/threonine kinase domain. In vitro, GEF domain 1(GEFD1) is specifically active on Rac1, while GEF domain 2 (GEFD2) targets RhoA. To determine whether Trio could activate Rac1 and RhoA in vivo, we measured the effect of Trio on Mitogen Activated Protein Kinase (MAPK) pathways and cytoskeletal rearrangements events mediated by the two GTPases. We show that: (i) the GEFD1 domain of Trio triggers the MAPK pathway leading to Jun kinase (JNK) activation and the production of membrane ruffles; (ii) co-expression of the TrioGEFD1 domain with a dominant-negative form of Rac blocked JNK induction, whereas a dominant-negative form of Cdc42 did not; (iii) a deletion mutant of TrioGEFD1 lacking a region important for exchange activity could not stimulate JNK activity; (iv) in contrast, the TrioGEFD2 domain does not stimulate JNK activity and induces the formation of stress fibers, as does activated RhoA; (v) furthermore, co-expression of both GEF domains induces simultaneously the formation of ruffles and stress fibers. Trio, therefore represents a unique member of the Rho-GEFs family possessing two functional domains of distinct specificities, that allow it to link Rho and Rac signaling pathway in vivo.