This study examined the regulation of glutamine synthetase protein levels, in response to changes in external glutamine concentration, in mouse C2C12 skeletal muscle cells. Glutamine, at concentrations as low as 0.25 mmol/L, downregulated endogenous and exogenous (plasmid encoded) glutamine synthetase with maximal effect at 2 mmol/L. Glutamine appears to act by changing the stability of the glutamine synthetase protein, and the effect was partially blocked by the proteasome inhibitor MG132. The addition of the glutamine structural analog and glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, in the presence or absence of glutamine, also resulted in low glutamine synthetase protein levels. Otherwise, the effect was specific for glutamine, and the only compounds able to mimic the effect of glutamine were amino acids, glutamate, alanine, and ornithine, which can be converted to glutamine. Other amino acids, analogs, and products of glutamine metabolism were without effect. Methionine sulfoximine, an inhibitor of glutamine synthetase, stabilized the protein and prevented the glutamine effect. Thus, in mouse C2C12 skeletal muscle cells, glutamine synthetase protein expression is regulated by glutamine through changes in the rate of degradation of the protein. The effect is specific to glutamine, which acts directly without requiring prior metabolism.