The product of the Klebsiella pneumoniae nitrogen regulatory gene ntrC has been purified and shown to be a dimeric protein of subunit molecular weight 54Kd, designated NtrC. In an in vitro coupled transcription-translation system NtrC inhibited expression from both the ntrBC and glnA promoters. NtrC bound to both of these ntr repressible promoters with equal affinity, but did not bind to the activatable nitrogen fixation promoters nifF or nifLA. NtrC makes contact with nucleotides flanking the -10 region of the glnA (RNA2) promoter at sequences homologous with the proposed consensus binding site.