During nitrogen-limited growth, transcription of glnA, which codes for glutamine synthetase, requires sigma 54-RNA polymerase and the phosphorylated from the nitrogen regulator I (NRI; also called NtrC). In cells in which the lac promoter controlled expression of the gene coding for NRI, increasing the intracellular concentration of NRI lowered the level of glutamine synthetase. The reduction in glutamine synthetase does not appear to result from the NRI-dependent sequestering of any protein that affects transcription of glnA. Our results also suggest that the negative effect of a high concentration of NRI on glnA expression is a major determinant of the level of glutamine synthetase activity in nitrogen-limited cells of a wild-type strain. We propose that the inhibition results from an impairment of the interaction between NRI-phosphate and RNA polymerase that stimulates glnA transcription. We discuss a model that can account for this reduction in glutamine synthetase.