High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase σ32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus σ32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli σ32 and cross-reacts with a monoclonal antibody to E. coli σ32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in σ32 levels after a temperature shift from 30 to 42°C similar to that described for E. coli. In addition, increasing the cellular content of σ32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30°C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and σ32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to σ32 expression in this organism. Both the rpoH gene and σ32 protein were expressed constitutively throughout the cell cycle at 30°C. The isolation of rpoH provides an important tool for future studies of the role of σ32 in the normal physiology of C. crescentus.