The rpoH (htpR) gene of Escherichia coli encodes a sigma factor which confers upon RNA polymerase the ability to recognize the promoters for genes responsive to the phenomenon termed the heat shock response. dnaA protein, a sequence-specific DNA binding protein, is required for initiation of chromosomal replication by binding to sites within the chromosomal origin. dnaA protein also autoregulates its expression by binding to a site in the dnaA promoter region. Two copies of the dnaA protein recognition sequence are present within the rpoH promoter region. Using filter binding assays, dnaA protein was observed to bind specifically to DNA fragments containing the rpoH promoter region with greater affinity than its binding to the dnaA promoter region. By contrast, reduced binding to a DNA fragment containing the lacUV5 promoter was observed. DNase I footprint analysis indicated that dnaA protein protected specific sites within the rpoH promoter region. The binding of dnaA protein to the rpoH promoter region resulted in transcriptional repression from two of the three promoters of the rpoH gene in vitro. Elevated levels of dnaA protein repressed transcription from these two rpoH promoters in vivo. These results indicate that dnaA protein regulates rpoH transcription to influence the expression of genes under rpoH control.