The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO₂, serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO₂, 3% H₂, 0.087 ppm O₂) and microaerophilic (1% CO₂, 3.48 ppm O₂) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO₂. In this study we identified CO₂ as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO₂, independent of O₂ levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO₂. Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO₂, indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO₂ may be an additional cue for borrelial host adaptation and gene regulation.