Activated platelets have been implicated in playing a major role in transfusion-related acute lung injury (TRALI), as platelets can trigger neutrophils, resulting in vascular damage. We hypothesized that binding of platelet CD40 ligand (CD40L) to endothelial CD40 is essential in the onset of TRALI. Mice were challenged with monoclonal major histocompatibility complex (MHC)-1 antibody which induced TRALI, evidenced by pulmonary oedema, accompanied by significantly elevated bronchoalveolar fluid (BALF) levels of total protein and elevated plasma levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) compared to infusion of isotype antibody (all Ps<0·05). Treatment with ciglitazone, which inhibits platelet CD40L expression, had no effect on pulmonary and systemic inflammation compared to controls. In addition, treatment with anti-CD40L antibody, which antagonizes all CD40-CD40L interactions, also did not abrogate the TRALI reaction. Furthermore, levels of soluble CD40L were measured in a cohort of cardiac surgery patients, who were followed prospectively for the onset of TRALI after transfusion. Plasma levels of sCD40L at baseline and at time of developing TRALI did not differ between TRALI patients and controls (transfused cardiac surgery patients not developing acute lung injury) (275±192 versus 258±346 and 93±82 versus 93±123pg/ml, respectively, not significant). In conclusion, these results do not support the idea that the CD40-CD40L interaction is involved in mediating TRALI.