Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoS_Bb) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoS_Bb recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoS_Bb dependent, while the ospE and flaB reporters were RpoS_Bb independent. To compare promoter recognition by RpoS_Bb with that of the prototype RpoS (RpoS_Ec), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoS_Ec the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoS_Bb under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoS_Ec and RpoS_Bb each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(−14) in the presumptive −10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoS_Bb as it is for RpoS_Ec; (ii) the nucleotide at the −15 position determines RpoS or σ⁷⁰ selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoS_Ec- or RpoS_Bb-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.