The promoter region(s) for the rRNA operons of Rhodobacter sphaeroides was identified. By utilizing both in vivo and in vitro techniques, the transcriptional start sites of all three operons were identified. Upstream of the transcriptional start, -10 and -35 promoter regions that bear little similarity to typical Escherichia coli promoter sequences were identified. In addition to the promoter sequences, probable Fis protein-binding sites were identified upstream of all three rRNA operons. Transcriptional fusions of the promoter regions from rrnA and rrnB were constructed by utilizing the reporter molecule encoded by xylE and analyzed under various growth conditions, in both a wild-type background and an rrnBC mutant background. Production of the xylE gene product (catechol 2,3-dioxygenase) was always greatest under photosynthetic growth conditions. However, the upstream region of rrnB, when fused with xylE, produced significantly more catechol 2,3-dioxygenase than did analogous regions of rrnA, suggesting that the promoters of the rrn operons differ in strength. These results were further confirmed by the study of mutant strains altered for the rrn operons either singly or in combination. Little or no expression of the xylE gene was manifest in E. coli when directed by rDNA sequences derived from R. sphaeroides.