Reactive oxygen species (ROS) produced during mitochondrial activity participate in the regulation of intracellular signaling pathways. However, there is only limited information concerning the role that ROS derived from the mitochondrial respiratory chain play in modulating neutrophil activity and participation in acute inflammatory processes. Because mitochondrial complex III is a major site of ROS formation, we examined whether selective complex III inhibition, through exposure of neutrophils to myxothiazol or antimycin A, would affect LPS-induced activation. Culture of neutrophils with antimycin A or myxothiazol resulted in increased intracellular levels of ROS, including superoxide and hydrogen peroxide (H2O2). Inhibition of complex III activity reduced LPS-induced degradation of IκB-α, nuclear accumulation of NF-κB, and proinflammatory cytokine production. The effects of antimycin A or myxothiazol appeared to be dependent on generation of H2O2 since addition of pegylated catalase to neutrophils restored LPS-mediated IκB-α degradation and production of proin-flammatory cytokines. Administration of myxothiazol to mice resulted in diminished mitochondrial complex III activity in the lungs and decreased severity of LPS-induced lung injury. These results indicate that inhibition of mitochondrial complex III diminishes Toll-like receptor 4-induced neutrophil activation through a mechanism dependent on H2O2 generation and also reduces the severity of lung injury due to LPS exposure, a pathophysiologic process in which neutrophils play a major role. Copyright © 2009 the American Physiological Society.