We studied the regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Increased enzyme activity was specific for glucocorticoids; other steroid hormones were essentially without effect. The induction of glutamine synthetase was selective, in that glutaminase activity was not induced by dexamethasone treatment of L6 cells. Thus, glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.