Background: The control of syphilis depends on screening of the population at risk and is usually performed using the Treponema pallidum particle agglutination test (TPPA). Outside Europe the rapid plasma reagin test (RPR) or venereal disease research laboratory test is most often used for screening purposes. Because of the drawbacks in current diagnostic procedures, ie, long turnaround time, the need is felt for a rapid and simple test that can potentially be performed on whole blood. Objective and study design: In this study a one- step immunochromatographic test (Biorapid Syphilis) and two ELISA, the Bioelisa Syphilis 3.0 and ETI-Treponema Plus, were evaluated. Methods: Serum samples were collected between February 2000 and May 2006 at the University Hospital in Maastricht, The Netherlands. 145 TPPA- positive sera, confirmed by fluorescent treponemal antibody absorption (FTA- Abs, treponemal test) and/or RPR (non-treponemal) were included. Furthermore, 41 sera from healthy controls and 144 TPPA- negative sera from controls with underlying conditions that might interfere with T pallidum serology were collected. Results: The sensitivity and specificity of the Biorapid Syphilis, Bioelisa Syphilis 3.0 and ETI- Treponema Plus were 92% and 79%, 100% and 100% and 100% and 100%, respectively, with our selected sera. Conclusions: The performance of both ELISA was excellent in our study and is favoured over the TPPA because of its ability to be run on an automated system. The sensitivity and specificity of the Biorapid Syphilis were considered too low to implement the test in a hospital laboratory in a developed country but it might be useful in primary healthcare settings in developing countries.