Intestinal epithelial cells (IECs) provide a physical and immunologic barrier against the enteric microbial flora. Toll-like receptors (TLRs), through interaction with conserved microbial patterns, activate inflammatory gene expression in cells of the innate immune system. Previous studies of expression and function of TLRs in IECs report varying results. Therefore, TLR expression was characterized in human and murine intestinal sections, and TLR function tested in an IEC line. TLR1, TLR2, TLR4 are co-expressed on a subpopulation of human and murine IECs that reside predominantly in the intestinal crypt and belong to the enteroendocrine lineage. An enteroendocrine cell (EEC) line demonstrates a similar expression pattern of TLRs as primary cells. The murine EEC line, STC-1, was activated with specific TLR ligands: lipopolysaccharide (LPS) or synthetic bacterial lipoprotein. In STC-1 cells stimulated with bacterial ligands, NF-B and MAP kinase activation is demonstrated. Furthermore, expression of TNF and MIP-2 are induced. Additionally, bacterial ligands induce expression of the anti-inflammatory gene TGF-. LPS triggers a calcium flux in STC-1 cells, resulting in a rapid increase in cholecystokinin secretion. Finally, conditioned media from STC-1 cells inhibit the production of NO and IL-12 p40 by activated macrophages. In conclusion, human and murine IECs that express TLRs belong to the enteroendocrine lineage. Using a murine EEC model, a broad range of functional effects of TLR activation is demonstrated. This study suggests a potential role for EECs in innate immune responses.