Increased lung vascular permeability is an important contributor to respiratory failure in acute lung injury (ALI). We found that a function-blocking antibody against the integrin αvβ5 prevented development of lung vascular permeability in two different models of ALI: ischemia-reperfusion in rats (mediated by vascular endothelial growth factor [VEGF]) and ventilation-induced lung injury (VILI) in mice (mediated, at least in part, by transforming growth factor-β [TGF-β]). Knockout mice homozygous for a null mutation of the integrin β5 subunit were also protected from lung vascular permeability in VILI. In pulmonary endothelial cells, both the genetic absence and blocking of αvβ5 prevented increases in monolayer permeability induced by VEGF, TGF-β, and thrombin. Furthermore, actin stress fiber formation induced by each of these agonists was attenuated by blocking αvβ5, suggesting that αvβ5 regulates induced pulmonary endothelial permeability by facilitating interactions with the actin cytoskeleton. These results identify integrin αvβ5 as a central regulator of increased pulmonary vascular permeability and a potentially attractive therapeutic target in ALI.