Pseudomonas aeruginosa, an opportunistic respiratory pathogen that infects the majority of patients with cystic fibrosis, initiates host inflammatory responses through interaction with airway epithelial cells. The Toll-like receptors (TLRs) are a family of pathogen pattern recognition receptors that play key roles in host innate immunity. In this study we aimed to determine whether TLRs mediate the interaction between P. aeruginosa and airway epithelial cells. Individual murine TLRs (TLR1 to TLR9) and dual combinations of these TLRs that activate an NF-kappaB-driven luciferase reporter in response to PAO1 were screened in HEK 293 cells. TLR5, TLR2, a combination of TLR1 and TLR2, or a combination of TLR2 and TLR6 responded to PAO1. Another P. aeruginosa strain, strain PAK, activated TLR5 similarly, while the isogenic flagellin-deficient strain PAK/fliC and the flagellum-free bacterium Haemophilus influenzae failed to activate TLR5. Reverse transcription-PCR was used to probe the presence of multiple TLRs (including TLR5) in primary human airway epithelial cells (HAECs). Immunostaining with TLR5 antibodies showed that TLR5 was expressed in HAECs and on the apical surface of the human trachea epithelium. In HAECs, PAO1, PAK, and Burkholderia cepacia, but not flagellin-deficient strain PAK/fliC or a B. cepacia fliC mutant, activated the NF-kappaB reporter. Dominant negative TLR5 specifically blocked the response to P. aeruginosa but not to the response to lipoteichoic acid, a specific ligand of TLR2. We also determined that MyD88, IRAK, TRAF6, and Toll-interacting protein (Tollip), but not TIRAP, were involved in the TLR-mediated response to P. aeruginosa in HAECs. These findings demonstrate that the airway epithelial receptor TLR5 senses P. aeruginosa through its flagellin protein, which may have an important role in the initiation of the host inflammatory reaction to clear the invading pathogen.