Lung endothelial damage is a characteristic morphological feature of ischemia‐reperfusion (I/R) injury, although the molecular steps involved in the loss of endothelial integrity are still poorly understood. We tested the hypothesis that the activation of vascular endothelial growth factor (VEGF) cell signaling would be responsible for the increase in lung vascular permeability seen early after the onset of I/R in rats. Furthermore, we hypothesized that the I/R‐induced pulmonary edema would be significantly attenuated in rats by the activation of the stress protein response. Pretreatment with Ad Flk‐1, an adenovirus encoding for the soluble VEGF receptor type II, prevented I/R‐mediated increase in lung vascular permeability in rats. Furthermore, the I/R‐induced lung injury was significantly decreased by prior activation of the stress protein response with geldanamycin or pyrrolidine dithiocarbamate. In vitro studies demonstrated that VEGF caused an increase in protein permeability across primary cultures of bovine macro‐ and microvascular lung endothelial cell monolayers that were associated with a phosphorylation of VE‐ and E‐cadherin and the formation of actin stress fibers. Activation of the stress protein response prevented the VEGF‐mediated changes in protein permeability across these cell monolayers and reduced the phosphorylation of VE‐and E‐cadherins, as well as the formation of actin stress fibers in these cells.—Godzich, M., Hodnett, M., Frank, J. A., Su, G., Pespeni, M., Angel, A., Howard, M. B., Matthay, M. A., Pitte, J. F. Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia‐reperfusion injury in rats. FASEB J. 20, E757–E768 (2006)