A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced ALI has been shown, however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species (ROS) and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on transwell inserts and incubated with wildtype and NADPH oxidase deficient AM under in the presence or absence of cytomix (equimolar TNF-, IL-1 and IFN-). Injury to the monolayers was assessed by trans-PMVEC Evans-Blue labelled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC co-cultures we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NAPDH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute PMVEC injury in our studies. From our findings we concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability.