ABSTRACT We have identified a second RpoH homolog, RpoH _II , in the α-proteobacterium Rhodobacter sphaeroides . Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoH _II belongs to a phylogenetically distinct group with RpoH orthologs from α-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoH _I , RpoH _II is able to complement the temperature-sensitive phenotype of an Escherichia coli σ ³² ( rpoH ) mutant. In addition, we show that recombinant RpoH _I and RpoH _II each transcribe two E. coli σ ³² -dependent promoters ( rpoD P _HS and dnaK P1) when reconstituted with E. coli core RNA polymerase. We observed differences, however, in the ability of each sigma factor to recognize six R. sphaeroides promoters ( cycA P1, groESL ₁ , rpoD P _HS , dnaK P1, hslO , and ecfE ), all of which resemble the E. coli σ ³² promoter consensus. While RpoH _I reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters, RpoH _II produced detectable transcripts from only four promoters ( cycA P1, groESL ₁ , hslO , and ecfE ). These results, in combination with previous work demonstrating that an RpoH _I mutant mounts a typical heat shock response, suggest that while RpoH _I and RpoH _II have redundant roles in response to heat, they may also have roles in response to other environmental stresses.