The mechanisms by which mediators and cytokines stimulate neutrophils to migrate across the lung epithelium are still unclear. We hypothesized that neutrophil transepithelial migration depends upon polarity of the epithelium. We therefore compared neutrophil migration through human lung Type II-like alveolar epithelial cell line (A549) monolayers grown on the upper versus lower surface of permeable filters to simulate apical-to-basal and basal-to-apical movement of neutrophils, respectively. The classic chemoattractants formyl-methionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8) induced equivalent neutrophil transepithelial migration in the apical-to-basal and basal-to-apical directions. However, the degree of neutrophil transepithelial migration was significantly greater in the basal-to-apical direction in response to either IL-1beta or tumor necrosis factor-alpha (TNF-alpha). Enhanced TNF-alpha-induced neutrophil migration through A549 monolayers in the basal-to-apical direction occurred regardless of whether the TNF-alpha was above or below the filter/monolayer complex. Actinomycin D pretreatment of A549 monolayers had no effect on FMLP-induced neutrophil transepithelial migration, but markedly (about 75%) inhibited both TNF-alpha- and IL-1beta-induced neutrophil transepithelial migration, regardless of monolayer orientation. Thus, in contrast to classic chemoattractants, IL-1beta and TNF-alpha induced greater neutrophil transepithelial migration in a basal-to-apical direction, and this occurred independently of the cytokine location, but depended upon intact metabolic capacity of the A549 cells. These data suggest that the mechanisms important for neutrophil transepithelial migration in response to classic chemoattractants differ from those important for migration in response to inflammatory cytokines.