We investigated the mechanism for the induction of a chemokine, IL-8, by bacterial flagellins in the human alveolar type II epithelial cell line, A549. Bacterial flagellin induced expression of IL-8 mRNA and protein in dose- and time-dependent manners. IL-8 expression was inhibited by nystatin (a lipid rafts inhibitor) but not by chlorpromazine (a clathrin-coated pits inhibitor). Interestingly, Toll-like receptor 5 (TLR5) recognizing flagellins was found in the intracellular compartment of A549 but rarely on the cell surface. Flagellin-induced IL-8 expression appears to be mediated through TLR5 as determined by in vitro transient transfection experiment in HEK-293 cells expressing TLR5 using a reporter gene construct containing IL-8 promoter. IL-8 expression was attenuated by inhibitors for protein kinase C (PKC) and mitogen-activated protein (MAP) kinases. Furthermore, NF-kappaB and NF-IL6 transcription factors played an important role in the flagellin-induced IL-8 gene expression in A549 cells. Collectively, these results suggest that flagellin-induced IL-8 expression requires formation of lipid rafts, intracellular TLR activation, and subsequent activation of PKC and MAP kinases leading to the activation of the transcription factors NF-kappaB and NF-IL6 in human alveolar type II epithelial cells.