Glutamine is important for growth, proliferation, and differentiation of the small intestine. Whether glutamine produced within intestinal epithelial cells is important for these processes is not known. We hypothesize that blockade of endogenous glutamine synthesis in rat intestinal crypt epithelial cells (IEC-6) results in a decrease in cell proliferation. IEC-6 cells were cultured in media containing 8 different concentrations of glutamine ranging from 0.02 to 8.06 mM glutamine, with or without the GS inhibitor, methionine sulfoximine (MS). Cell number, total protein (mg per plate), total DNA (μg per plate), and tritiated-thymidine incorporation (per μg of DNA) varied in a positive dose-dependent manner with media glutamine concentration. In the presence of MS, cell proliferation was reduced in cultures grown in 2mM or less glutamine (p < 0.05), but not at the supraphysiologic concentrations of 4.06 or 8.06 mM glutamine. To determine whether IEC-6 cells upregulate GS activity in response to glutamine deficiency, we compared GS activities of post-confluent cultures fed media containing no supplemental glutamine for 24, 96, and 240 hrs to control cultures grown in media containing 4.06 mM glutamine. Glutamine synthetase activities after 24, 96, and 240 hrs of glutamine deprivation were 89, 105, and 93% greater than that of control cultures fed 4.06 mM glutamine (p < 0.05). We also demonstrated that cultures in which proliferation is inhibited by exposure to MS recover following withdrawal of the inhibitor. Glutamine synthetase, an enzyme previously given little attention in the small intestine, appears to be critical for proliferation of crypt cells. We speculate that agents which affect the activity of this enzyme could influence proliferation of the intestinal epithelium.