The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ−/−) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients ( kf) or wet-to-dry weight ratios between PKCδ+/+ and PKCδ−/− mice. However, at 24 h after exposure to LPS, the kf values were significantly higher in lungs isolated from PKCδ+/+ than PKCδ−/− mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ+/+ mice displayed increased protein and cell content compared with LPS-exposed PKCδ−/− mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ+/+ mouse lung parenchyma relative to PKCδ−/− mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased kf lung values in PKCδ+/+ mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ−/− mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.