Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS- S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.