Alterations in endothelial permeability are a defining feature of diverse processes including arteriosclerosis, inflammation, ischemia/reperfusion injury, angiogenesis, pulmonary edema in acute lung injury and adult respiratory distress syndrome. Endothelial monolayer permeability increases as a result of both disruption of endothelial cell-cell contacts and EC contraction. Disruption of endothelial cell-cell junctions occurs concomitantly with the redistribution and tyrosine phosphorylation of the VE-cadherin-containing adherens junction (AJ) protein complexes. Little is known about mechanisms of how endothelial permeability is regulated. Reactive oxygen species (ROS) including superoxide (O2-) and hydrogen peroxide (H2O2) generated by activated polymorphonuclear leukocyte (PMNs) and endothelial cells (ECs) impair endothelial barrier integrity by promoting loss of cell-cell adhesions and reorganization of actin cytoskeleton. These responses are involved in promoting transendothelial migration of PMNs and endothelial permeability. Major source of ROS in PMNs and ECs is NADPH oxidase. Phagocyte NADPH oxidase consists of membrane-bound gp91phox and p22hox as well as cytosolic components such as p47phox, p67phox and small GTPase Rac. Recently, several novel homologues of gp91phox (Nox2) of NADPH oxidase (Nox) have been cloned in non-phagocytic cells. In ECs Nox1, Nox2, Nox4 and Nox5 are functionally expressed. NADPH oxidase in ECs is activated by inflammatory cytokines, thrombogenic agents, growth factors, G-protein coupled receptor agonists and shear stress. ROS derived from NADPH oxidase function as signaling molecules to activate various redox signaling pathways through modulating activity of kinases and phosphatases, which may contribute to increase in endothelial permeability. Understanding mechanisms by which ROS regulate endothelial permeability is important for the development of novel therapeutic approaches against various diseases such as inflammation, atherogenesis and acute lung injury.