The Rhodobacter capsulatus NtrC (RcNtrC) is a novel bacterial enhancer-binding protein that can activate transcription in an RpoN-independent manner. The nifR3-ntrB-ntrC operon in R. capsulatus codes for the nitrogen-sensing two component regulators RcNtrB and RcNtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes. A nifR3-lacZ translational fusion showed 20-fold lower levels in ntrC mutants compared to the wildtype. In contrast, an operon fusion (i.e. ntrC-lacZ) was expressed at the same levels irrespective of nitrogen and ntrC. Western immunoblot analysis confirmed the nitrogen- and NtrC-dependent expression of NifR3 and constitutive production of RcNtrC. A single transcription start site of the operon is located directly upstream of the nifR3 open reading frame, a result consistent with genetic studies and promoter deletion analysis of the ntrC-lacZ fusion. The levels of nifR3 transcript are equivalent in wild type and NtrC- strains grown under various nitrogen sources. Evidence was obtained that supports a model involving direct control of NifR3 expression by RcNtrC. The purified RcNtrC protein was shown to bind to the nifR3 promoter region in vitro at two sets of tandem binding sites located from +8 to -99 nucleotides relative to the transcriptional start site. Deletion analysis demonstrated that the upstream tandem sites are essential for nitrogen- and NtrC-dependent production of NifR3 in vivo, but are not necessary for nifR3 transcription. Results of these studies suggest that RcNtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA. This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA, and anfA) in R. capsulatus which are transcriptionally activated by RcNtrC at tandem binding sites centered greater than 100 bps upstream of the transcriptional start site.