The generation of reactive oxygen species (ROS) plays a major role in endothelial signaling and function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase (Nox) family of proteins, Nox1, Nox2, Nox4 and Nox5, are major contributors of ROS. Excess generation of ROS contributes to the development and progression of vascular disease. While hyperoxia stimulates ROS production through Nox proteins, hypoxia appears to involve mitochondrial electron transport in the generation of superoxide. ROS generated from Nox proteins and mitochondria are important for oxygen sensing mechanisms. Physiological concentrations of ROS function as signaling molecule in the endothelium; however, excess ROS production leads to pathological disorders like inflammation, atherosclerosis, and lung injury. Regulation of Nox proteins is unclear; however, antioxidants, MAP Kinases, STATs, and Nrf2 regulate Nox under normal physiological and pathological conditions. Studies related to redox regulation of Nox should provide a better understanding of ROS and its role in the pathophysiology of vascular diseases.