The mechanisms involved in the restoration of endothelial cell junctions subsequent to barrier disruption remain unclear. It is known that formation of adherens junctions (AJs) affects cytoskeletal actin arrangement and that Rho GTPases regulate the state of actin polymerization. In the present study, we examined the role of the Rho GTPases, Rho, Rac, and Cdc42 in the reannealing of AJs. We studied the response to thrombin, which increases endothelial permeability through disassembly of AJs, followed by recovery of barrier function through junctional reannealing within 2 hours. Cdc42 was activated late, at approximately 1 hour after thrombin exposure, concurrent with its translocation from the cytoplasm to the membrane. Activation and translocation of Cdc42 preceded the reformation of AJs. Expression of the dnCdc42 mutant (N17Cdc42) significantly delayed the reformation of the VE-cadherin-containing AJs and restoration of endothelial barrier function. We also studied the lung microcirculation to address the in vivo relevance of Cdc42 signaling in barrier restoration. N17Cdc42 expression in the mouse lung endothelium markedly attenuated the endothelial barrier recovery after the permeability increase induced by activation of the thrombin receptor protease-activated receptor-1. These findings demonstrate the critical function of Cdc42 in restoring AJ-dependent, endothelial cell homotypic adhesion and barrier function. The delayed activation of Cdc42 represents a negative-feedback mechanism that signals AJ reassembly after the increase in endothelial permeability induced by inflammatory mediators such as thrombin.