Epithelial cells and lymphocytes, including γδ and αβ T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of γδ and αβ T cell responses. When highly purified CD3+ IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-β, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both γδ and αβ T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokines in γδ and αβ IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses. Further, mIEC did not inhibit splenic αβ T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial γδ and αβ T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.