Leukotriene B4 (LTB(4)) is a potent lipid mediator of inflammation that possesses antiviral activities. Here we provide evidence that LTB(4)-mediated defense against in vitro cytomegalovirus (CMV) infection of human leukocytes involves activation of the high-affinity LTB(4) receptor (BLT1) and neutrophil degranulation. Treatment of CMV-infected peripheral blood leukocytes with LTB(4) (10 nM) leads to a significant reduction in viral titers. This activity involves neutrophil activation through the BLT1 receptor, because no reduction in viral titers was observed after neutrophil depletion from cellular preparation or when leukocytes were pretreated with the BLT1 antagonist U75,302. Direct stimulation of neutrophils with LTB(4) (in the presence or absence of CMV) leads to the release of myeloperoxidase, alpha-defensins, eosinophil-derived neurotoxin, and the human cathelicidin LL-37 in a BLT1-dependent manner. LTB(4) does not act exclusively on the secretion of preformed antimicrobial peptides, but also acts on the synthesis of selected peptides as reflected by the increase in transcriptional levels of eosinophil-derived neurotoxin (EDN) and LL-37 in LTB(4)-treated neutrophils. Treatment of cell cultures with neutralizing antibodies directed against alpha-defensins, EDN, and LL-37 significantly reduces the antiviral effect of LTB(4), suggesting that LTB(4) may act through the release of antimicrobial peptides. Ex vivo experiments using LTB(4)-treated neutrophils from peritoneal washing of wild-type and BLT1 knockout mice further supported the role played by antimicrobial peptides in LTB(4)-mediated antiviral activity toward CMV. These results provide evidence of a mechanism by which LTB(4) induces host defense against viral infection.