Soluble antigens of Treponema pallidum were examined by two dimensional immunoelectrophoresis against antisera from infected or artificially immunized rabbits. Concentrated suspensions of intact cells did not release antigens after storage at 4 degrees, incubation at 37 degrees, or vortex mixing. Antigens were released after disintegration of treponemes by ultrasonic vibration, or by treatment with non-ionic or anionic detergents. An antigenic component of sonicated treponemes, present in both the non-pathogenic, cultivable Reiter treponeme and T. Pallidum, was identified as axial filament. The combination of antibody with unfixed whole organisms was monitored by an indirect fluorescent antibody method, and whereas antibody did not combine with intact organisms, detergent-treated organisms were highly reactive. Immune electron microscopy showed that whereas in intact treponemes, axial filaments were unable to combine with antibody, detergent treatment allowed access to axial filaments by antibody. In intact treponemes the axial filaments are thought to be located beneath the outer membrane, which may thus comprise the postulated antigenically inert outer layer.