The development of IgM and IgG antibody responses to antigenic molecules of Treponema pallidum was examined in intratesticularly and intradermally infected rabbits by use of immunofluorescence (IF) and Western blotting techniques. IgM antibody was first detectable on day 6 following intratesticular infection and reached maximal IF titers during the period of clinical orchitis (days 10-17). IgG reached peak IF titers in the resolution period following clearance of most organisms from the testes (after day 17). Initial IgM antibody responses on day 6 were directed against the 46-, 43-, and 37-kdalton molecules, with reactivity to the 14- and 12-kdalton molecules appearing on day 10. IgG antibody, which was first apparent in the Western blot on day 6, was directed against the 37-kdalton molecule. IgG antibody to the 46-kdalton molecule was first apparent on day 13, and the full spectrum of 22 molecules was recognized on day 31. Curative therapy of intradermally infected rabbits one week after infection resulted in lower IgG antibody titers but did not abrogate the development of humoral responses to all of the major antigens.