The cellular and biochemical mechanisms leading to acute lung injury (ALI) and subsequent multiple organ failure are only partially understood. To study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of ALI, we used a murine experimental model of ALI induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration, as shown by immunofluorescence and protein leakage into the alveolar space were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase (5-LO) pathway, and transgenic mice deficient in 5-LO were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared with sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-Lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-LO, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of ALI induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury. © 2012 by the Shock Society.