Hyperoxia-induced acute lung injury has been used for many years as a model of oxidative stress mimicking clinical acute lung injury and the acute respiratory distress syndrome. Excess quantities of reactive oxygen species (ROS) are responsible for oxidative stress-induced lung injury. ROS are produced by mitochondrial chain transport, but also by NADPH oxidase (NOX) family members. Although NOX1 and NOX2 are expressed in the lungs, their precise function has not been determined until now.To determine whether NOX1 and NOX2 contribute in vivo to hyperoxia-induced acute lung injury.Wild-type and NOX1- and NOX2-deficient mice, as well as primary lung epithelial and endothelial cells, were exposed to room air or 100% O(2) for 72 hours.Lung injury was significantly prevented in NOX1-deficient mice, but not in NOX2-deficient mice. Hyperoxia-dependent ROS production was strongly reduced in lung sections, in isolated epithelial type II cells, and lung endothelial cells from NOX1-deficient mice. Concomitantly, lung cell death in situ and in primary cells was markedly decreased in NOX1-deficient mice. In wild-type mice, hyperoxia led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two mitogen-activated protein kinases involved in cell death signaling, and to caspase-3 activation. In NOX1-deficient mice, JNK phosphorylation was blunted, and ERK phosphorylation and caspase-3 activation were decreased.NOX1 is an important contributor to ROS production and cell death of the alveolocapillary barrier during hyperoxia and is an upstream actor in oxidative stress-induced acute lung injury involving JNK and ERK pathways in mice.