To determine the role of phosphoinositide 3-OH kinase (PI3K) pathways in the acute vascular permeability increase associated with ventilator-induced lung injury, we ventilated isolated perfused lungs and intact C57BL/6 mice with low and high peak inflation pressures (PIP). In isolated lungs, filtration coefficients (K_f) increased significantly after ventilation at 30 cmH₂O (high PIP) for successive periods of 15, 30 (4.1-fold), and 50 (5.4-fold) min. Pretreatment with 50 μM of the PI3K inhibitor, LY-294002, or 20 μM PP2, a Src kinase inhibitor, significantly attenuated the increase in K_f, whereas 10 μM Akt inhibitor IV significantly augmented the increased K_f. There were no significant differences in K_f or lung wet-to-dry weight (W/D) ratios between groups ventilated with 9 cmH₂O PIP (low PIP), with or without inhibitor treatment. Total lung β-catenin was unchanged in any low PIP isolated lung group, but Akt inhibition during high PIP ventilation significantly decreased total β-catenin by 86%. Ventilation of intact mice with 55 cmH₂O PIP for up to 60 min also increased lung vascular permeability, indicated by increases in lung lavage albumin concentration and lung W/D ratios. In these lungs, tyrosine phosphorylation of β-catenin and serine/threonine phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), and ERK1/2 increased significantly with peak effects at 60 min. Thus mechanical stress activation of PI3K and Src may increase lung vascular permeability through tyrosine phosphorylation, but simultaneous activation of the PI3K-Akt-GSK3β pathway tends to limit this permeability response, possibly by preserving cellular β-catenin.