Enteroaggregative Escherichia coli (EAEC) causes watery diarrhoea that is often mildly inflammatory. Previous studies have reported that the flagellin of EAEC induces IL-8 from intestinal epithelial cells (IECs) in culture. To characterize more fully the inflammatory response to EAEC, we infected IECs with EAEC prototype strain 042 and assessed cellular responses by macroarray and reverse transcriptase polymerase chain reaction (RT-PCR). Genes upregulated in 042-infected non-polarized T84 cells included IL-8, IL-6, TNF-alpha, GRO-alpha, GRO-gamma, ICAM-1, GM-CSF and IL-1alpha. RT-PCR analyses performed with cDNA from T84 and HT-29 cells infected with an aflagellar mutant (042fliC) suggested that these responses were primarily mediated by flagellin. To better reproduce the conditions of the infection for this non-invasive pathogen, we assessed the responses of polarized IECs to strain 042 infection. As expected, 042 induced IL-8 production from both polarized T84 and HT-29 cells. However, significant IL-8 secretion was induced in polarized T84 cells infected with 042fliC, suggesting that a factor other than flagellin contributes to inflammation in this model. This non-flagellar IL-8 response required expression of the aggregative adherence fimbria (AAF) adhesin, and was related to the presence of the minor fimbria-associated protein AafB. Our data suggest that multiple factors contribute to EAEC-induced inflammation, and further characterization of the nature of EAEC proinflammatory factors will greatly advance our understanding of this emerging pathogen.