Heterotrimeric G-proteins of the Galpha12/13 family activate Rho GTPase through the guanine nucleotide exchange factor p115RhoGEF. Because Rho activation is also dependent on protein kinase Calpha (PKCalpha), we addressed the possibility that PKCalpha can also induce Rho activation secondary to the phosphorylation of p115RhoGEF. Studies were made using human umbilical vein endothelial cells in which we addressed the mechanisms of PKCalpha-induced Rho activation and its consequences on actin cytoskeletal changes. We observed that PKCalpha associated with p115RhoGEF within 1 min of thrombin stimulation and p115RhoGEF phosphorylation was dependent on PKCalpha. Inhibition of PKCalpha-dependent p115RhoGEF phosphorylation prevented the thrombin-induced Rho activation, indicating that the response occurred downstream of PKCalpha phosphorylation of p115RhoGEF. The regulator of G-protein signaling domain of p115RhoGEF, a GTPase activating protein for G12/13, also prevented thrombin-induced Rho activation, indicating the parallel requirement of G12/13 in signaling Rho activation via p115RhoGEF. These data demonstrate a pathway of Rho activation involving PKCalpha-dependent phosphorylation of p115RhoGEF. Thus, Rho activation in endothelial cells and the subsequent actin cytoskeletal re-arrangement require the cooperative interaction of both G12/13 and PKCalpha pathways that converge at p115RhoGEF.