Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47 phox and gp91 phox ) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47 phox؊/؊ and gp91 phox؊/؊ mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47 phox؊/؊ and gp91 phox؊/؊ mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-B pathway in response to CS exposure in p47 phox؊/؊ and gp91 phox؊/؊ mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-B inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.