The ontogeny of the IgG response in rabbits with experimental syphilis to individual polypeptides of Treponema pallidum, Nichols strain, was examined. The polypeptides of motile, virulent T. pallidum, purified from host tissue by Percoll density gradient centrifugation, were separated on SDS-polyacrylamide gels and were electrophoretically transferred to a solid-phase matrix of nitrocellulose for antigen analysis. Serum from rabbits early in infection at day 3 post-infection showed a weak but detectable IgG response to two polypeptides of 60,000 and 46,000 m.w. This was followed on days 11, 17, and 19 by an apparent quantitative increase in antibody to other treponemal protein antigens. By day 19, 21 of the 22 detectable polypeptides could be identified. A similar set of antigens was detected by serum from patients with human secondary and early latent syphilis. A close correlation was found between the presence of IgG antibody to T. pallidum polypeptides at day 9 and 1, 4.5, 13.5, and 17 mo post-infection and the immune status of the rabbit to symptomatic reinfection. Serum from rabbits that were partially immune to challenge at day 9 detected three polypeptides of 60,000, 46,000, and 36,000 daltons. By 1 mo post-infection, at a time when a more complete immunity had developed, the number of detectable antigens increased to 21 polypeptides ranging in m.w. from 94,000 to 14,400 daltons. IgG antibody to 22 treponemal antigens persisted in animals that were solidly immune to symptomatic reinfection at 3, 4.5, 13.5, and 17 mo post-infection. Serum neutralizing activity was not demonstrable at day 9 or 1 mo post-infection, however, but was present at 3, 4.5, 13.5, and 17 mo. The results suggest that after intratesticular challenge a vigorous IgG response to T. pallidum polypeptides can be detected. The potential role of humoral immune mechanisms in the development and maintenance of immunity is discussed.