The development of genetic systems for B. burgdorferi has allowed identification of several regulatory and structural genes involved in the ability of this bacterium to colonize and disseminate in its arthropod and mammalian hosts. Progress has been slow as a result of the complex biology of this pathogen, and its genetic distance from Gram-positive and Gram-negative bacteria has impeded easy adaptation to B. burgdorferi of genetic tools developed for other bacteria. We have repeatedly encountered these issues in our attempts to use molecular genetic tools to analyze the role of the members of the paralogous bmp gene family in the biology and virulence of B. burgdorferi. For example, an attempt to characterize the role of bmpC in virulence using a bmpC deletion mutant of an infectious B. burgdorferi 297 strain and an extrachromosomally complemented derivative of this mutant was only partially successful. While deletion of bmpC did not decrease infectivity in mice, it significantly decreased B. burgdorferi DNA in joints and arthritis. Extrachromosomal complementation of this null mutation reduced infectivity and did not restore tissue pathogenicity. To overcome these problems, we have adapted the Tet system of controlled gene expression developed for other bacteria and eukaryotic cells to manipulate gene expression in B. burgdorferi. In a two plasmid Tet system, where one plasmid contains the TetR repressor fused to the flaB promoter and the other plasmid contains a hybrid borrelial promoter with a tet operator (Ptetl-1), expression of the gene fused to the Ptetl-1 promoter (truncated BmpA, green fluorescent protein) is regulated by anhydrotetracycline in a concentration dependent manner over a wide range of concentrations. Because the two plasmid Tet system has some inherent instability manifested by escape from TetR repression and potential plasmid loss requiring continuous antibiotic selection, we constructed a B. burgdorferi strain containing the flaB/tetR fusion inserted in a nonessential gene (luxS) and designed a new hybrid promoter containing two instead of one tet operators (Ptetl-2). The functionality of this Tet system was demonstrated by manipulating expression of the B. burgdorferi bmpA gene. These findings strongly suggest that the Tet system can be used in B. burgdorferi to manipulate gene expression and open the door to manipulating gene expression by antisense RNA technology and gene fusions that will permit isolation of conditional lethal mutants in B. burgdorferi.