Human intestinal epithelial cells up-regulate the expression of an inflammatory gene program in response to infection with a spectrum of different strains of enteroinvasive bacteria. The conserved nature of this program suggested that diverse signals, which are activated by enteroinvasive bacteria, can be integrated into a common signaling pathway that activates a set of proinflammatory genes in infected host cells. Human intestinal epithelial cell lines, HT-29, Caco-2, and T84, were infected with invasive bacteria that use different strategies to induce their uptake and have different intracellular localizations (ie, Salmonella dublin, enteroinvasive Escherichia coli, or Yersinia enterocolitica). Infection with each of these bacteria resulted in the activation of TNF receptor associated factors, two recently described serine kinases, IκB kinase (IKK) α and IKKβ, and increased NF-κB DNA binding activity. This was paralleled by partial degradation of IκBα and IκBε in bacteria-infected Caco-2 cells. Mutant proteins that act as superrepressors of IKKβ and IκBα inhibited the up-regulated transcription and expression of downstream targets genes of NF-κB that are key components of the epithelial inflammatory gene program (ie, IL-8, growth-related oncogene-α, monocyte chemoattractant protein-1, TNF-α, cyclooxygenase-2, nitric oxide synthase-2, ICAM-1) activated by those enteroinvasive bacteria. These studies position NF-κB as a central regulator of the epithelial cell innate immune response to infection with enteroinvasive bacteria.