A molecular characterization of the polypeptide antigens of Treponema pallidum reactive with sera from patients with different stages of syphilis is described. Polypeptides of motile, virulent T. pallidum, purified from rabbit testes, were separated on SDS polyacrylamide gels and electrophoretically transferred to nitrocellulose for antigenic analysis ("Western blotting"). Serum IgG from uninfected individuals reacts weakly with three polypeptides of 45,000, 33,000, and 30,000 m.w. In this study patients with primary syphilis have IgM antibody, and all patients with syphilis have IgG antibody to at least four polypeptides of 45,000, 33,000, 30,000, and 15,500 m.w. Antibody to polypeptides of 42,000 and 16,500 m.w. appear to be markers for nonprimary syphilis. These six polypeptides have been termed the major antigenic proteins (MAP) of T. pallidum. Those patients studied with secondary and early latent syphilis acquire antibody to a set of 16 additional polypeptide antigens. Those patients studied with late latent or late syphilis have antibody to a much smaller set of five or four antigens, respectively, in addition to MAP. The results suggest that a correlation exists between acquisition of antibody and the development of "chancre immunity." Additionally, the loss of antibody that characterizes late latent and late syphilis may be associated with the potential development of destructive late syphilis.