We developed a single-plasmid-based regulatable protein expression system for Borrelia burgdorferi. Expression of a target gene is driven by P(ost), a hybrid B. burgdorferi ospA-tetO promoter, from a recombinant B. burgdorferi plasmid constitutively expressing TetR. The system was tested using the green fluorescent protein (GFP) as a reporter. Under noninducing conditions, recombinant B. burgdorferi cells were nonfluorescent, no GFP protein was detected, and residual, small amounts of transcript were detectable only by reverse transcription-PCR but not by Northern blot hybridization. Upon induction with anhydrotetracycline, increasing levels of GFP transcript, protein, and fluorescence were observed. This tight and titratable promoter system will be invaluable for the study of essential borrelial proteins. Since target protein, operator, and repressor are carried by a single plasmid, the system's application is independent of a particular strain background.