RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein G␣ 13 stimulates the guanine nucleotide exchange factor (GEF) activity of RHRhoGEFs, leading to activation of RhoA. The mechanism by which G␣ 13 stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in G␣ 13 that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of G␣ 13 to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by G␣ 13 and not through its interaction with a secondary binding site. A crystal structure of G␣ 13 bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a G␣ 13 -G␣ i1 chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by G␣ 13 .Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of ␣, , and ␥ subunits, act as molecular switches that cycle between an inactive, GDP-bound state and an active, GTP-bound state upon stimulation of G protein-coupled receptors (1). Once activated, the GTP-bound G␣ subunit dissociates from the G␥ dimer, both of which regulate the activity of multiple intracellular effectors to elicit cellular responses. The duration of signaling mediated by G␣ is dictated by the lifetime of bound GTP. All G␣ subunits have intrinsic GTPase activity that hydrolyzes GTP to GDP, and this rate of hydrolysis is enhanced by GAPs, 2 such as RGS proteins (2), which bind the switch regions of activated G␣ subunits, stabilize the transition state for GTP hydrolysis, and thus accelerate the hydrolysis of GTP to GDP (3, 4). Once in the GDP-bound state, G␣ subunits reassociate with G␥ and are capable of initiating another round of signaling.G␣ 13 is one of two members of the G 12 family of G proteins (5) and has been implicated in regulating multiple cellular processes that depend on activation of the monomeric GTPase RhoA, including gene transcription, embryogenesis, and rearrangement of the actin cytoskeleton (6 -8). A direct link between G␣ 13 and RhoA was established with the discovery of p115RhoGEF, the founding member of a family of RhoA-specific GEFs containing an RH domain in their N termini (RHRhoGEFs) (9). Together with p115RhoGEF, PDZ-RhoGEF and LARG constitute the RH-RhoGEF family in mammals (10, 11). The RH domain of p115RhoGEF has low sequence similarity to canonical RGS proteins but nevertheless functions as a GAP for G␣ 13 (12). Although the structure of the RH domain of p115RhoGEF is similar to that of other RGS proteins (13), additional elements flanking the RGS box are required for GAP activity (14) and stability of the isolated domain (15). In addition to its RH domain, p115RhoGEF also contains the tandem DH/PH domains characteristic of Dbl family RhoGEFs. The nucleoti...