Transcription of the Bacillus subtilis gene coding of glutamine synthetase (glnA) is regulated by the nitrogen source. The glnA gene lies in an operon in which it is preceded by an open reading frame with the potential to encode a polypeptide of approximately 16,000 Mr. We have now shown that this open reading frame is utilized in vivo, that its product (GlnR) acts as a diffusible, negative regulator of gln transcription, and that GlnR is likely to be a DNA-binding protein. Certain mutations in glnR, including a large, in-frame deletion and a start codon mutation, led to high-level constitutivity of the operon; other mutations caused low-level constitutivity. These latter mutations, which affected the C terminus of GlnR, seemed to disrupt response to the nitrogen source without eliminating the ability of GlnR to bind to DNA. Wild-type GlnR by itself, however, did not impose nitrogen-dependent regulation; such regulation also required the product of glnA. A model is presented in which glutamine synthetase monitors the availability of nitrogen and imposes negative regulation by interaction with or modification of GlnR.