Description: Objective— Antibodies against human neutrophil antigen-3a (HNA-3a) located on choline transporter-like protein 2 induce severe transfusion-related acute lung injury (TRALI). This study aims to identify the mechanism implicated in anti–HNA-3a-mediated TRALI. Approach and Results— Our analysis shows that anti–HNA-3a recognizes 2 choline transporter-like protein 2 isoforms (P1 and P2) on human microvascular endothelial cells from lung blood vessels but reacts only with the P1 isoform on neutrophils. Direct treatment of HNA-3a–positive endothelial cells with anti–HNA-3a, but not with anti–HNA-3b, leads to reactive oxygen species production, increased albumin influx, and decreased endothelial resistance associated with the formation of actin stress filaments and loosening of junctional vascular endothelium-cadherin. In a novel in vivo mouse model, TRALI was documented by significant increase in lung water content, albumin concentration, and neutrophil numbers in the bronchoalveolar lavage on injection of human anti–HNA-3a in lipopolysaccharides-treated, as well as nontreated mice. Interestingly, although neutrophil depletion alleviated severity of lung injury, it failed to prevent TRALI in this model. Infusion of anti–HNA-3a F(ab') 2 fragments caused moderate TRALI. Finally, mice lacking nicotinamide adenine dinucleotide phosphate oxidase (NOX2 y/ - ) were protected from anti–HNA-3a-mediated TRALI. Conclusions— These data demonstrate the initiation of endothelial barrier dysfunction in vitro and in vivo by direct binding of anti–HNA-3a on endothelial cells. It seems, however, that the presence of neutrophils aggravates barrier dysfunction. This novel mechanism of TRALI primarily mediated by endothelial cell dysfunction via choline transporter-like protein 2 may help to define new treatment strategies to decrease TRALI-related mortality.