Increased endothelial permeability is frequently associated with opening of interendothelial junctions that are mainly regulated by VE-cadherin/b-catenin complex. We recently showed that thrombin-mediated barrier function decrease is due to an endocytotic process involving a novel functional protein complex consisting of the epidermal growth factor receptor pathway substrate 15 (Eps15) and caveolin-1. This complex associates with b-catenin and following thrombin stimulation the Eps15/caveolin-1 disconnects b-catenin from the VE- cadherin/catenin complex leading to opening of interendothelial junctions as shown by coimmunoprecipitation and immunofluorescence studies. Rho-GTPases have been indicated to be critical involved in controlling interendothelial barrier function. Here we show that expression of dominant negative (d.n.) RhoA in endothelial cells completely blocks the barrier function decrease by inhibition of Eps15/caveolin-1 mediated disconnection and endocytosis of b-catenin. In contrast neither d.n. Rac1 nor d.n. Cdc42 were able to block endocytosis of the Ep15/caveolin-1/b-catenin complex. The data show that RhoA activation is required to open interendothelial junctions in response to thrombin by activating endocytosis.