Intestinal intraepithelial lymphocytes (IELs) are major effector cells in the gut mucosal immune system, and are phenotypically distinct from thymic and peripheral T cells. Although nutritional supplementation with glutamine affects the intestinal immune response, it remains unclear whether this is a direct effect via the IEL-derived cytokines. This study examined changes in IEL-derived cytokine production following treatment with glutamine in vitro. Murine IELs were purified and activated with PMA plus ionomycin, and then cultured in the presence of various glutamine concentrations. IEL-derived cytokines were measured using a cytometric bead array (CBA) system, and IEL subsets were analyzed by flow cytometry. Treatment with glutamine increased the production of IL-2 and IFN-gamma from IELs in the presence of PMA plus ionomycin, but had no effect on TNFalpha, IL-4, or IL-5 production. Treatment with alanine or glucose had no regulatory effect on IEL-derived cytokines. Glutamine therefore had a direct effect on the production of selected IEL-derived Th1-cytokines, and enteral supplementation with glutamine may influence the intestinal immune responses mediated by IELs.