Signaling specificity of Rho GTPase pathways is achieved in part by selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. For example, Trio, GEF-H1, and Tiam1 are a subset of GEFs that specifically activate Rac1 but not the closely related Cdc42. The Rac1 specificity of these GEFs appears to be governed by Rac1-GEF binding interaction. To understand the detailed mechanism underlying the GEF specificity issue, we have analyzed a panel of chimeras made between Rac1 and Cdc42 and examined a series of point mutants of Rac1 made at the switch I, switch II, and β2/β3regions for their ability to interact with and to be activated by the GEFs. The results reveal that Rac1 residues of both the switch I and switch II regions are involved in GEF docking and GEF-mediated nucleotide disruption, because mutation of Asp38, Asn39, Gln61, Tyr64, or Arg66/Leu67into Ala results in the loss of GEF binding, whereas mutation at Tyr32, Asp65, or Leu70/Ser71leads to the loss of GEF catalysis while retaining the binding capability. The region between amino acids 53–72 of Rac1 is required for specific recognition and activation by the GEFs, and Trp56in β3appears to be the critical determinant. Introduction of Trp56to Cdc42 renders it fully responsive to the Rac-specific GEFin vitroand in cells. Further, a polypeptide derived from the β3region of Rac1 including the Trp56residue serves as a specific inhibitor for Rac1 interaction with the GEFs. Taken together, these results indicate that Trp56is the necessary and sufficient determinant of Rac1 for discrimination by the subset of Rac1-specific GEFs and suggest that a compound mimicking Trp56action could be explored as an interfering reagent specifically targeting Rac1 activation.