We addressed the in vivo role of phosphotidylinositol 3-kinase (PI3K) in signaling the sequestration of polymorphonuclear leukocytes (PMN) in lungs and in the mechanism of inflammatory lung vascular injury. Studies were made in mice with deletion of the p110 catalytic subunit of PI3K (PI3K^-/-mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal (i.p.) E. coli challenge. PMN infiltration in the lung interstitium of PI3K^-/- mice as assessed morphometrically was increased 100% over controls within 1 hr after bacterial challenge. PI3K^-/- mice also developed a greater increase in lung microvascular permeability after E. coli challenge resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K^-/- mice was associated with increased expression of the PMN adhesive proteins CD47 and ₃ integrins. We observed increased association of CD47 and ₃ integrins with the extracellular matrix protein vitronectin in lungs of PI3K^-/- mice after E. coli challenge. PMNs obtained from these mice also showed increased ₃ integrin expression and augmented 3 integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K in negatively regulating the expression of CD47 and ₃ integrins in gram negative sepsis. PI3K activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to the expression of CD47 and ₃ integrins. Therefore, the level of PI3K activation may be an important determinant of PMN-dependent lung vascular injury.