Transcription of the Rhodobacter sphaeroides cytochrome c2 gene (cycA) is negatively regulated by both the presence of oxygen and intermediates in tetrapyrrole biosynthesis. A mutation responsible for uncoupling cycA transcription from tetrapyrrole availability was localized to a gene (chrR) that encodes a 357-amino-acid protein. Analysis of a defined chrR null mutation indicated that this protein positively regulated cycA transcription. From this and other results, it appeared that the positive action of ChrR on cycA transcription is blocked by altering the availability of either heme or some intermediate in tetrapyrrole biosynthesis. A single missense mutation which substitutes an Arg for a Cys at residue 182 of ChrR (C182R) was shown to be necessary and sufficient for the increased cycA transcription seen in the mutant strain Chr4. Thus, it appears that this C182R substitution generated an altered-function form of ChrR. In addition, by analyzing cycA transcription in delta ChrR strains, we showed that ChrR was not required for increased cycA transcription under anaerobic conditions. Instead, our results indicated that ChrR and the response regulator PrrA (J. M. Eraso and S. Kaplan, J. Bacteriol. 176:32-43, 1994) functioned independently at the upstream cycA promoter that is activated under anaerobic conditions.