Molecular analysis of the humoral response to pathogen-specific polypeptides was done using sera from patients at different stages of syphilis and sera from Treponema pallidum-infected rabbits and guinea pigs collected at various times after infection. The sera were examined by ELISA, fluorescent treponemal antibody absorption test (FTA-ABS), and immunoblot before and after sequential adsorption with cyanogen bromide-activated Sepharose 4B coupled individually to five sonicated nonpathogenic treponemes. Guinea pig antisera were also adsorbed with rabbit proteins. After adsorption, sera from all three species reacted neither by ELISA nor by immunoblot with nonpathogenic treponemes, nor did guinea pig sera react with rabbit proteins. Regardless of the species of treponema used for initial adsorption, none by itself could effectively remove all cross-reactivities. Nine pathogen-specific polypeptides (15, 17, 33, 37, 39, 43, 45, 47, 97 kDa), eight (15-47 kDa) known as integral membrane or endoflagellar components, were commonly recognized by the tree species when chancre immunity developed. The substantial reduction in immune reactivity after adsorption suggests that most of these peptides consist of specific and common treponemal epitopes. Throughout the various stages of the human disease, pathogen-specific antibodies were exclusively of the IgG isotype.