Outer surface lipoprotein (Osp) C is a virulence factor required for transmission of the Lyme disease agent, Borrelia burgdorferi. We have constructed an inducible promoter system to study the function and regulation of OspC by integrating regulatory elements from the Escherichia coli lac operon into the B. burgdorferi genome. An inducible promoter (flacp) was constructed by inserting a synthetic lac operator sequence between the transcriptional start site and the ribosomal binding site of the B. burgdorferi flgB promoter; flacp was then used to replace the native ospC and rpoS promoters in B. burgdorferi derivatives that constitutively express the E. coli Lac repressor protein (LacI). In vitro, the expression of ospC and rpoS from flacp was dependent on the inducer isopropyl β-D-thiogalactopyranoside and was unaffected by temperature or pH, conditions commonly used to mimic different aspects of the B. burgdorferi life cycle. Our results suggest that OspC is essential immediately upon injection into a mouse and OspC expression must be maintained during the early stages of infection. In addition, the mouse infectivity experiment indicates that this system can be used to regulate B. burgdorferi genes in vivo, within the context of an experimental tick-mouse infectious cycle. RpoS is an alternative sigma factor that is required for ospC transcription. However, the role of other temperature-dependent factors has not previously been addressed. Our results with the inducible rpoS strain demonstrate that RpoS alone is sufficient to activate OspC expression, even at 23°C. This is the first functional inducible promoter system developed for use in B. burgdorferi and, for the first time, will provide researchers with the ability to artificially regulate the expression of genes in this pathogenic spirochaete.