Glutamine synthetase transcipt quantities and enzyme activities in lung and skeletal muscle are elevated by glucocorticoids during stress and result in release of glutamine into the circulation for utilization primarily by the small intestine. The small intestine is presently recognized for its capacity to metabolize glutamine rather than the ability to synthesize glutamine. Data from our laboratory support a much more important role for glutamine synthesized by small intestinal crypt cells. This study examines the influence of DEX on steady state levels of glutamine synthetase mRNA and enzyme activity of rat intestinal crypt epithelial cells (IEC-6). We hypothesize that DEX upregulates expression of GS message and enzyme activity. We also hypothesize that DEX treatment causes an increase in IEC-6 cell proliferation. Post-confluent IEC-6 cells, cultured in DMEM containing 4 mM glutamine, were exposed to 10-9, 10-7, or 10-5 M DEX for 24 h. Following treatment with DEX, cultures were processed for measurement of GS activity and protein, and messenger RNA was isolated for analysis by RNA gel and dot blots. Steady state levels of GS mRNA increased by 256 and 383% in cultures treated with 10-7 and 10-5 M DEX, respectively (p < 0.05). GS activities of cultures treated with 10-9, 10-7, or 10-5 M DEX increased by 203, 500, and 559%, respectively (p < 0.05). Treatment with 10-7 M DEX results in a 28% increase in 3H-thymidine incorporation (p < 0.05). These data demonstrate that glucocorticoids regulate GS expression of rat crypt epithelial cells by increasing the production of GS transcripts and enzyme activity. The increase in glutamine synthesizing capacity that occurs following treatment with DEX is concurrent with an increase in cell proliferation. These data suggest that upregulation of the glutamine synthesizing capacity of intestinal crypt cells may be important during stress.