The TprI antigen of Treponema pallidum subsp. pallidum is a putative virulence factor predicted to be located in the outer membrane of the syphilis spirochete. In this study, we analyzed the immune response against TprI and its subunits in sera collected both from rabbits experimentally infected with the Nichols strain and from patients with syphilis, showing a different pattern of reactivity toward the antigen in these two groups of samples. The protective ability of recombinant TprI and its hypothetical outer membrane location were also investigated. Although no rabbit was protected after challenge, immunoelectron microscopy results, to be further investigated, were compatible with the outer membrane location of the antigen.