Metabolite control of glutamine synthetase expression (by glutamine) was studied in L6 skeletal muscle cells. Depletion of glutamine from the culture medium for 24 hours resulted in a 3-4-fold increase in glutamine synthetase activity. This effect was blocked by cycloheximide but not by actinomycin D. Addition of glutamine to L6 cells maintained in glutamine-free medium caused a rapid return of glutamine synthetase activity to the control level. As reported, dexamethasone caused a striking increase in the glutamine synthetase mRNA level. In contrast, neither depletion nor addition of glutamine caused a change in the glutamine synthetase mRNA level. Therefore, regulation of glutamine synthetase by glutamine is exerted at a post-transcriptional level.