Spirochetes that share pathogen-restricted antigens with Treponema pallidum subsp. pallidum have been identified in dental plaque and diseased gingival tissues, but it is not known whether these spirochetes possess virulence characteristics. In this study, plaque spirochetes were able to transmigrate a tissue barrier in vitro and were identified on the other side by using monoclonal antibodies specific for pathogen-restricted determinants from T. pallidum subsp. pallidum. This invasive capability is shared with T. pallidum subsp. pallidum, but cultured oral and intestinal treponemes did not perforate the tissue barrier. Cocultures indicated that invasive treponemes do not create opportunities for cultivable oral treponemes to cross the barrier. These findings indicate that gingival tissues may be a port of entry for previously unrecognized invasive spirochetes in humans.