Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (P(ospF)) and ospE (P(ospE)) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by sigma(70). Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to P(ospF), P(ospE), or two hybrid promoters in which the -10 regions of P(ospF) and P(ospE) were switched [P(ospF ) ((E - 10)) and P(ospE) ((F - 10)) respectively]. We found that the P(ospF)-10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while sigma(70) specificity for P(ospE) is dependent on elements outside of the -10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to sigma(70). Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have -10 regions virtually identical to that of P(ospF). Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the P(ospF)-10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the -10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding.