The initial response to infection is recruitment of acute inflammatory cells to the involved site. Interleukin (IL)-8 is the prototypical effector molecule for this process. Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-kappaB. Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora. We therefore sought to investigate NF-kappaB activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E. coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-kappaB factor is causally linked to IL-8 production. We report herein that NF-kappaB is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E. coli lipopolysaccharide. Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship. Furthermore, deletion of specific EPEC virulence genes abrogates the NF-kappaB-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response. These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-kappaB, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs. nonpathogens.