To study the different patterns of Treponema pallidum distribution in primary and secondary syphilis, 34 biopsy specimens of 8 patients with primary and 26 with secondary syphilis were assessed. Histopathological features, silver stain, and immunohistochemical T pallidum polyclonal antibody expression were investigated. The number and distribution of spirochetes were evaluated, and ultrastructural studies were performed. Spirochetes were identified with Warthin-Starry stain in 17 specimens (4/8 primary and 13/26 secondary syphilis), whereas immunohistochemical analysis disclosed spirochetes in 29 (8/8 primary and 21/26 secondary syphilis). In secondary syphilis, an epitheliotropic pattern characterized by abundant spirochetes in the lower mucosa/epidermis in an intercellular distribution was observed. In contrast, primary syphilis exhibited a mixed epitheliotropic and vasculotropic pattern further manifested by treponemes surrounding the vascular walls. These differences were statistically significant. Ultrastructural examination confirmed these results. Immunohistochemistry shows greater sensitivity when compared with Warthin-Starry staining. The immunohistochemical pattern of T pallidum distribution may permit the diagnostic differentiation of primary from secondary syphilis.