The Dbl-like guanine nucleotide exchange factor (GEF) Lbc oncoprotein specifically activates the small GTP-binding protein Rho in mammalian fibroblasts to induce transformation and actin stress fiber formation, whereas another Dbl-related molecule, Cdc24, stimulates guanine nucleotide exchange of the Rho family GTPase Cdc42 to elicit effects on both gene induction and actin-based cytoskeleton change in Saccharomyces cerevisiae. To understand the mechanism of these functional interactions, we have taken a biochemical approach to probe the sites on Rho and Cdc42 that are involved in coupling to their respective GEFs, the Lbc and Cdc24 proteins. Point mutations in the switch II region of the small G-proteins, many of which would affect the interaction with GEF in the case of Ras, or a mutation in the switch I region that was identified as a contact site between Rab3A and Rab GEF had little effect on RhoA or Cdc42Hs with regard to the ability to interact with Lbc or Cdc24, suggesting that there exists a unique mechanism of regulation of the Rho family proteins by their GEFs. Analysis of a panel of chimeras made between RhoA and Cdc42Hs, which all maintained the ability to respond to Dbl, their mutual GEF, and to GTPase-activating protein, revealed that at least two distinct sites in each of the GTPases are required for activation by the respective GEFs. Further site-directed mutagenesis studies showed that the conserved residue Tyr32 in the putative effector region of both GTPases (numbered by Cdc42Hs) is critical for binding of the GEFs and that specific recognition for Lbc or Cdc24 is achieved at least in part through residues Lys27 of Rho and Gln116 of Cdc42. Moreover, the loss of GEF responsiveness of a RhoA mutation (D76Q) was found to be caused by the impaired GEF catalysis, not by a change in the GEF binding affinity. Together, these results indicate that multiple sites of the Rho GTPases are involved in the regulation by GEFs, contributing to GEF binding or GEF catalysis, and raise the possibility that activation of each Rho family G-protein by a specific GEF may engage in a distinct mechanism.