The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A fulllength cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.