The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.