Toll-like receptors (TLRs) activate anti-microbial gene expression in response to detection of specific bacterial products. Relatively little is known about TLR5, the only TLR thought to be preferentially expressed by epithelial cells, beyond that it confers activation of the transcription factor NF-B in a MyD-88 dependent manner in response to flagellin. Since TLRs, in general, are also thought to signal through members of the MAP kinase family we examined MAP kinase activation (via examining its phosphorylation status), and its subsequent role in expression of the chemokine IL-8 in polarized intestinal epithelia. Flagellin, like other pro-inflammatory stimuli (TNF, S. typhimurium), activated p38 MAPK in a TLR5-dependent manner whereas aflagellate bacteria or the growth factor EGF did not activate this kinase. While ERK 1,2 was also observed to be activated in response to flagellin, its activation was not restricted to pro-inflammatory stimuli as it was also potently activated by aflagellate bacteria (S. typhimurium or E. coli) and EGF (neither of which activate NF-B in these cells). Pharmacological inhibition of p38 MAPK (by SB-203580) potently (IC50=10nM) reduced expression of IL-8 protein (maximal inhibition 75%) but had no effect on NF-B activation, only slightly attenuated up-regulation of IL-8 mRNA levels in response to flagellin, and did not affect IL-8 mRNA stability. Together, these results indicate that epithelial TLR5 mediates p38 MAPK activation and subsequently regulates flagellin-induced IL-8 expression independent of NF-B likely by influencing IL-8 mRNA translation.