Bacterial core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Escherichia coli encodes seven σ factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of σ³², the heat-shock σ factor, and estimate that there are 120–150 σ³² promoters in total. Unexpectedly, 25% of these σ³² targets are located within coding regions, suggesting novel regulatory roles for σ³². The majority of σ³² promoter targets overlap with those of σ⁷⁰, the housekeeping σ factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPσ⁷⁰ and RNAPσ³² initiating transcription in vitro with similar efficiency and from identical positions. σ^E-regulated promoters also overlap extensively with those for σ⁷⁰. These results suggest that extensive functional overlap between σ factors is an important phenomenon.