Treponema pallidum, the etiological agent of syphilis, is characterized by a paucity of surface exposed outer membrane proteins and a high content of cytoplasma membrane associated lipoproteins. At all stages of infection intense antibody responses against lipoproteins are detectable. In order to provide antigens for syphilis diagnosis the highly immunogenic lipoproteins TpN17, TpN29-35 (TpD), TpN44.5 (TmpA), TpN47, and TpN35 (TmpC) and the membrane protein TpN39 (BMP) were cloned. Insertion of PCR amplified DNA into an E. coli expression vector resulted in high level expression of antigens. N-terminal hexahistidine sequence allowed efficient purification of fusion proteins by metal chelate affinity chromatography. The recombinant antigens were tested in enzyme-linked immunosorbent assays. TpN17, TpN47, and TpN44.5 antigens showed high antibody titers. Assays with the three antigens combined resulted in a further improvement of diagnostic sensitivity in comparison with single antigens. Antibodies were found in 17 of 18 patients in all stages of syphilis, whereas 42 normal human sera were nonreactive. No cross-reactivity was detected in 24 sera of patients with Lyme borreliosis.