Ribosomal RNAs in E. coli are transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the major E. coli rRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for the rrnB and rrnD fragments and less pronounced for the rrnA and rrnE operons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.