BACKGROUND Transfusion‐related acute lung injury (TRALI) is a major complication of hemotherapy that may occur after the transfusion of any blood type component. Several clinical reports have suggested the presence of anti‐HLA antibodies in the blood product. This study sought to examine the role of anti‐HLA‐A2 antibodies in polymorphonuclear neutrophil (PMN) activation and thus in endothelial permeability. STUDY DESIGN AND METHODS PMN activation was assessed by both nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activity and reactive oxygen species (ROS) production. A coculture assay of EA.hy926 endothelial cells with PMNs or differentiated‐PLB‐985 cells, a model of neutrophil‐like cells, was performed to estimate the impact of ROS on endothelial permeability. RESULTS Anti‐HLA‐A2 antibodies significantly increased PMN activation, with subsequent endothelial dysfunction. Phagocyte NADPH oxidase (NOX2) activity was shown to be involved in this process and ROS themselves were demonstrated to induce VE‐cadherin cleavage and endothelial permeability. CONCLUSION Our data may support the existence of a critical anti‐HLA‐A2 antibody threshold for PMN activation, with NOX2 activity and subsequent endothelial permeability in the two‐hit model of TRALI.