Epithelial cells constitute a physical barrier to sexual transmission of HIV, but are also a source of cytokines that could alter infection efficiency. We studied HIV infection of the human colonic epithelial cell line HCT116, which is a model for differentiation of intestinal mucosal epithelium. Differentiated HCT116 cells had increased expression of cell surface C-X-C chemokine receptor type-4 (CXCR4) that mediated HIV entry, despite the apparent absence of cell surface CD4. HIV infection in differentiated HCT116 cells increased the levels of IL-1alpha, and IFN-alpha mRNA even though only 1% of cells had integrated provirus. The inefficient, CXCR4-mediated infection of differentiated HCT116 cells supports the view that epithelial cells are a barrier and not a portal for HIV transmission. However, low level infection of epithelial cells could trigger the release of cytokines that indirectly increase the transmission rate.