Leukotriene B4 (LTB4) is a potent inflammatory mediator involved in the pathogenesis of many pulmonary diseases. Although the neutrophil is the predominant source of LTB4, other cells can also interact with neutrophils and increase LTB4 formation. In this study, we investigated whether human neutrophil-airway epithelial cell interactions can increase LTB4 formation. Neutrophils were cocultured with transformed airway epithelial cells (9HTEo- cells), and LTB4 and leukotriene A4 (LTA4) degradation product release was measured by high-performance liquid chromatography and ultraviolet spectrometry. When stimulated with the calcium ionophore A-23187, neutrophil-9HTEo- cell cocultures released more LTB4 and less LTA4 degradation products in a time- and dose-related manner than did neutrophils alone. This increase in LTB4 release involved the metabolism of neutrophil-derived LTA4 to LTB4 by 9HTEo- cells and was affected by the neutrophil-to-epithelial cell ratio. Enhanced LTB4 release required proximity between neutrophils and 9HTEo- cells but not specific cell-cell adhesion. Our data demonstrate that human neutrophil-airway epithelial cell interactions can increase LTB4 formation through transcellular arachidonic acid metabolism.