Leukotriene B(4) (LTB(4)) is a lipid mediator of inflammation that was recently shown to exert antiviral activities. In this study, we demonstrate that the release of antimicrobial proteins by neutrophils contribute to an early host defense against influenza virus infection in vitro as well as in vivo. Daily i.v. treatments with LTB(4) lead to a significant decrease in lung viral loads at day 5 postinfection in mice infected with influenza A virus compared with the placebo-treated group. This reduction in viral load was not present in mice deficient in the high-affinity LTB(4) receptor. Viral clearance in lungs was associated with up-regulated presence of antimicrobial peptides such as beta-defensin-3, members of the mouse eosinophil-related RNase family, and the mouse cathelicidin-related antimicrobial peptide. Our results also indicate that neutrophils are important in the antiviral effect of LTB(4). Viral loads in neutrophil-depleted mice were not diminished by LTB(4) administration, and a substantial reduction in the presence of murine cathelicidin-related antimicrobial peptide and the murine eosinophil-related RNase family in lung tissue was observed. Moreover, in vitro treatment of human neutrophil cultures with LTB(4) led rapidly to the secretion of the human cathelicidin LL-37 and eosinophil-derived neurotoxin, known as antiviral peptides. Pretreatment of cell cultures with specific LTB(4) receptor antagonists clearly demonstrate the implication of the high-affinity LTB(4) receptor in the LTB(4)-mediated activity. Together, these results demonstrate the importance of neutrophils and the secretion of antimicrobial peptides during the early immune response mediated by LTB(4) against a viral pathogen.