Neutrophil recruitment from the blood stream into the airways is one of the important features of many inflammatory disorders in the lung. In this process, neutrophils migrate first from the blood across the ECs in the apical-to-basolateral direction into the tissues and then across airway epithelium in the basolateral-to-apical direction into the lung lumen. We investigated and compared the different mechanisms of neutrophil transmigration in vitro across monolayers of these two cell types in both directions. Neutrophil migration induced by chemoattractants was stronger across resting EC than across resting epithelial cells when both cell types were growing on top of the filters (i.e., migration in the apical-to-basolateral direction). Much higher neutrophil transepithelial migration was found in the physiological, basolateral-to-apical direction (cells hanging underneath the filters) than in the opposite direction across either resting or IL-1 beta-activated epithelial cells. In contrast, no significant difference was observed with EC under these conditions. After a 4-h IL-1 beta activation, neutrophil migration across EC was dependent on IL-8 and PAF. However, the migration across epithelial cells relied, to a greater extent, on IL-8 production, and not on PAF. The adhesion molecule ICAM-1 contributed much less to neutrophil migration across epithelium than that across endothelium. Our study provides evidence of different mechanisms of neutrophil transmigration across monolayers of EC and epithelial cells in vitro, indicating that different processes control neutrophil transmigration across EC and epithelial cells in vivo.