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 IB-, nuclear accumulation of NF-B, 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 IB- degradation and production of proinflammatory 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 TLR4 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.