ABSTRACT Treponema pallidum and closely related bacteria are the causative agents of syphilis, yaws, bejel (endemic syphilis), and pinta. Syphilis alone has an estimated prevalence of 18 million cases worldwide, and in 2016 the total number of reported syphilis cases in the United States was the highest recorded since 1993. One of the barriers to research on T. pallidum has been the inability to culture the spirochete continuously in vitro; it is one of the few major bacterial pathogens (along with Mycobacterium leprae) with this distinction. In the preliminary data for this project, we report the consistent, long-term multiplication of T. pallidum subsp. pallidum, the causative agent of syphilis, in a tissue culture system. The culture system consists of co-incubation of T. pallidum with rabbit epithelial cells in a modified tissue culture medium under a microaerobic atmosphere containing 1.5% O2 and 5% CO2. Exponential multiplication of T. pallidum has been obtained for over 100 days in ongoing experiments, with excellent retention of viability (as measured by motility) and infectivity (examined by experimental infection of rabbits). Optimal replication of T. pallidum occurs with subculture at 6 to 7 day intervals and periodic medium exchange, which are thought to maintain near-homeostatic conditions and to minimize entrance of the cultures into the stationary phase of growth. In this project we propose to continue and expand studies on the factors affecting T. pallidum multiplication in this system and to explore its utility in T. pallidum research, as exemplified by mutational analysis. The specific aims are to 1) determine the optimal conditions for continuous growth of T. pallidum subspecies and strains in the tissue culture system; 2) utilize the tissue culture system to define the nutritional requirements with the purpose of achieving culture of T. pallidum under axenic conditions (i.e. without mammalian cells); and 3) examine the feasibility of genetic analysis of in vitro cultured T. pallidum through chemical mutagenesis, transformation with oligonucleotides, shuttle vectors and suicide plasmids, and transposon mutagenesis. The further confirmation and development of the T. pallidum in vitro culture system will likely facilitate many aspects of T. pallidum research, including studies of physiology, structure, antimicrobial susceptibility, pathogenesis, and host immunity.