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Intestinal epithelial cells have receptors that recognize bacterial antigens and in some circumstances are actively involved in bacterial internalization. To test the hypothesis that intestinal epithelial cells possess bactericidal capabilities, the bactericidal activity of two intestinal cell lines (IEC-18 and Caco-2) was measured using Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli as test organisms. The relative bactericidal efficacy of these two intestinal cell lines to kill these bacteria was compared against neutrophils (PMN) using a standard in vitro bactericidal assay. The IEC-18 and Caco-2 cells as well as the PMNs killed S. aureus and P. aeruginosa but not E. coli (p < .05). In fact, when tested in serum-free medium, the IEC-18 and Caco-2 cells killed a greater percentage of bacteria than the PMNs (p < .05). The addition of the antioxidant, superoxide dismutase, significantly reversed the bactericidal activity of both Caco-2 cells and neutrophils for P. aeruginosa and S. aureus, while catalase had no effect. Nitric oxide inhibition by NG-nitro-L-arginine methyl ester (L-NAME) had no effect on bactericidal activity of Caco-2 cells. These results indicate that intestinal epithelial cells can kill certain strains of bacteria and may function as “nonprofessional” phagocytes. Additionally, the mechanisms involved in the killing of P. aeruginosa and S. aureus by the Caco-2 and IEC-18 cells appear similar to the PMNs to the extent that bactericidal activity appeared to be oxidant-mediated but not nitric oxide-mediated in both the Caco-2 cell line and in the neutrophils.