The Bacillus subtilis glnR gene (part of the glnRA operon) encodes a 135-amino-acid (aa) repressor, GlnR, that regulates glnRA transcription in response to nitrogen levels in the growth medium. Two glnR mutants unable to repress under nitrogen excess conditions were obtained by mutagenesis. Lesions were found at Leu77 and Ala80, aa that lie within a region (between aa 59-83) thought to form the alpha-helix-turn-alpha-helix (HTH) motif common among a class of regulatory proteins. Alteration of Gly72 by site-directed mutagenesis also affected regulation, suggesting that aa within the putative HTH region are critical for GlnR function and may be involved in DNA binding. However, other replacements within the aa 59-83 sequence failed to support the HTH structure proposed for this region. Mutations within the C-terminal region of GlnR were also found to affect regulation. Introduction of an ochre stop codon at aa 110, 116, 123 and 129 resulted in the production of truncated proteins that were constitutively repressed, strongly suggesting that a signal recognition site residues within the last seven aa of GlnR. Substituting Asp129 with Asn led to loss of repression, indicating that Asp129 may be directly involved in interacting with either positive or negative effector molecules, or is a target for post-translational modification.