The semaphorins are a large family of secreted and cell surface proteins that provide attractive and repulsive cues for axon guidance during neuronal development. Semaphorins share a conserved NH(2)-terminal Sema domain with their receptors, the plexins, which mediate neuronal cell adhesion, axon guidance, and maintenance of established neuronal pathways in the adult. Both semaphorins and plexins share structural homology with the extracellular domain of c-Met, a member of the scatter factor family of receptors. However, the highly conserved cytoplasmic region of plexins has no homology with the c-Met tyrosine kinase or with any other known protein. Using a recently developed antibody and RNA analysis, we found that high levels of plexin-B1 are expressed in endothelial cells. Whereas c-Met, with which plexin-B1 can interact, is known to be a potent promoter of angiogenesis, the effects of semaphorin-mediated plexin activation in endothelial cells are still poorly understood. Here, we examined the role of plexin-B1 activation in angiogenesis using a purified, secreted form of its ligand, Semaphorin 4D (Sema4D). Sema4D potently induced chemotaxis and tubulogenesis in endothelial cells and enhanced blood vessel formation in an in vivo mouse model. Interestingly, responses to Sema4D did not require c-Met activation. Instead, the use of chimeric plexin-B1 receptors, Rho inhibitors, and lentiviral gene delivery of interfering molecules revealed that these proangiogenic effects are dependent on a COOH-terminal PDZ-binding motif of plexin-B1, which binds two guanine nucleotide exchange factors for the small GTPase Rho, PDZ-RhoGEF and LARG, and are mediated by the activation of Rho-initiated pathways.