In this study, we show that stimulation of human airway epithelial cells (HAECs) by Pseudomonas aeruginosa strain PAO1 induces time- and dose-dependent activation of p38 mitogen-activated protein kinase (MAPK). Activated p38 MAPK stayed in the cytoplasm instead of translocating to the nucleus, as shown by cellular fractionation. p38 MAPK was activated when HAECs were incubated with P. aeruginosa strain PAK and Burkholderia cepacia, while little activation was observed with the isogenic flagellin-free strains PAK/fliC and B. cepacia BC/fliC. The presence of Toll-like receptor 5 (TLR5) in 293 cells mediated PAO1-dependent activation of p38 MAPK, and in HAECs p38 MAPK activation was blocked by the overexpression of a dominant negative TLR5. Two inhibitors of p38 MAPK, SB202190 and SB203580, significantly attenuated PAO1-dependent expression of an NF-kappaB-dependent luciferase reporter gene, suggesting that p38 MAPK activation is required for full activation of NF-kappaB-dependent signaling. Microarray analysis of NF-kappaB target genes revealed up-regulation of multiple genes by PAO1 in HAECs. Reverse transcription-PCR and protein expression analysis were used to show that up-regulation of NF-kappaB-dependent genes induced by PAO1, such as the genes encoding Cox-2 and interleukin-8, was attenuated by SB203580. These results demonstrate a role for p38 MAPK signaling in gene regulation in response to P. aeruginosa via TLR5.