Although acute lung injury (ALI) is an important problem in humans, its pathogenesis is poorly understood. Airway instillation of bacterial LPS, a known complement activator, represents a frequently used model of ALI. In the present study, pathways in the immunopathogenesis of ALI were evaluated. ALI was induced in wild-type, C3(-/-), and C5(-/-) mice by airway deposition of LPS. To assess the relevant inflammatory mediators, bronchoalveolar lavage fluids were evaluated by ELISA analyses and various neutralizing Abs and receptor antagonists were administered in vivo. LPS-induced ALI was neutrophil-dependent, but it was not associated with generation of C5a in the lung and was independent of C3, C5, or C5a. Instead, LPS injury was associated with robust generation of macrophage migration inhibitory factor (MIF), leukotriene B(4) (LTB4), and high mobility group box 1 protein (HMGB1) and required engagement of receptors for both MIF and LTB4. Neutralization of MIF or blockade of the MIF receptor and/or LTB4 receptor resulted in protection from LPS-induced ALI. These findings indicate that the MIF and LTB4 mediator pathways are involved in the immunopathogenesis of LPS-induced experimental ALI. Most strikingly, complement activation does not contribute to the development of ALI in the LPS model.