Interleukin (IL)-1β has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1β-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1β production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the αvβ6 integrin and/or transforming growth factor-β attenuated the IL-1β-induced ALI. The results of in vitro studies indicated that IL-1β caused the activation of transforming growth factor-β via RhoA/αvβ6 integrin-dependent mechanisms and the inhibition of the αvβ6 integrin and/or transforming growth factor-β signaling completely blocked the IL-1β-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1β increased protein permeability across lung endothelial cell monolayers via RhoA- and αvβ5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the αvβ5 and αvβ6 integrins had an additive protective effect against IL-1β-induced ALI. In summary, these results demonstrate a critical role for the αvβ5/β6 integrins in mediating the IL-1β-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI. © 2008 American Heart Association, Inc.