Plexins are the first known transmembrane receptors that interact directly with small GTPases. On binding to certain Rho-family GTPases, the receptor regulates the remodeling of the actin cytoskeleton and alters cell movement in response to semaphorin guidance cues. In a joint solution NMR spectroscopy and x-ray crystallographic study we characterize a 110-residue cytoplasmic independent folding domain of plexin-B1 that directly binds three Rho-family GTPases, Rac1, Rnd1 and RhoD. The NMR data show that, surprisingly, the CRIB-like motif of plexin-B1 is not involved in this interaction. Instead, all three GTPases interact with the same region, -strands 3 and 4 and a short a-helical segment of the plexin domain. The 2.0 Å resolution x-ray structure shows that these segments are brought together by the tertiary structure of the ubiquitin-like fold. In the crystal, the protein is dimerized with C2 symmetry through a 4-stranded antiparallel -sheet that is formed outside the fold by a long loop between the monomers. This region is adjacent to the GTPase binding motifs identified by NMR. Destabilization of the dimer in solution by binding of any one of the three GTPases suggests a model for receptor regulation that involves bidirectional signaling. The model implies a multifunctional role for the GTPase-plexin interaction that includes conformational change and a localization of active receptors in the signaling mechanism.