When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT-PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian-like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb-infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out-compete for binding. bba66 promoter mapping revealed putative sigma70 and sigmaS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the -35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either sigmaN or sigmaS resulted in loss of BBA66. Promoter-GFP fusion analysis indicated that the sigma70 and/or sigmaS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.