ABSTRACT In the gammaproteobacteria the RpoH regulon is often equated with the stress response, as the regulon contains many of the genes that encode what have been termed heat shock proteins that deal with the presence of damaged proteins. However, the betaproteobacteria primarily utilize the HrcA repressor protein to control genes involved in the stress response. We used genome-wide transcriptional profiling to compare the RpoH regulon and stress response of Neisseria gonorrhoeae , a member of the betaproteobacteria. To identify the members of the RpoH regulon, a plasmid-borne copy of the rpoH gene was overexpressed during exponential-phase growth at 37°C. This resulted in increased expression of 12 genes, many of which encode proteins that are involved in the stress response in other species. The putative promoter regions of many of these up-regulated genes contain a consensus RpoH binding site similar to that of Escherichia coli . Thus, it appears that unlike other members of the betaproteobacteria, N. gonorrhoeae utilizes RpoH, and not an HrcA homolog, to regulate the stress response. In N. gonorrhoeae exposed to 42°C for 10 min, we observed a much broader transcriptional response involving 37 differentially expressed genes. Genes that are apparently not part of the RpoH regulon showed increased transcription during heat shock. A total of 13 genes were also down-regulated. From these results we concluded that although RpoH acts as the major regulator of protein homeostasis, N. gonorrhoeae has additional means of responding to temperature stress.