Bacterial flagellin is recognized by Toll-like receptor (TLR5) and activates NF-kappa B which leads to the induction of proinflammatory gene expression. Salmonella expresses two flagellin proteins, FliC and FljB. We purified FliC and FljB and examined the ability of the Salmonella flagellins to activate the NF-kappa B transcription factor in human embryonic kidney cells. We found that FliC and FljB as purified proteins possessed a comparable specific activity for activation of NF-kappa B-dependent gene expression in HEK293 cells. We also determined the ability of UV-inactivated bacteria, both wild-type and fliC and fljB mutant strains, to activate NF-kappa B. Wild-type flic(+)/fljB(+) Salmonella and the fliC(+)/fljB(-) mutant strain were robust activators, whereas the fliC(-)/fljB(+) and flhC(-) mutant strains were very poor activators. The NF-kappa B activation capacity of bacterial strains correlated with their flagellin expression level. Finally, Salmonella cell wall-associated polymeric flagellin displayed greatly reduced ability to activate NF-kappa B compared to purified monomeric flagellin. (c) 2007 Elsevier Inc. All rights reserved.