This retrospective study on syphilis screening at the sexually transmitted infection (STI) unit of a University Department emphasizes the necessity of a treponemal-specific test as the appropriate screening test. The Venereal Disease Research Laboratory (VDRL) test for syphilis screening may, under certain circumstances, yield positive results in patients not infected with Treponema pallidum, a phenomenon referred to as biological false-positive (BFP) VDRL test. The aim of this study was to determine the frequency of BFP tests in a large sample of sera. In this retrospective study, we analysed the results of parallel VDRL and T. pallidum haemagglutination (TPHA) testing of a total of 514,940 blood samples obtained from patients at the Vienna General Hospital between January 1988 and November 1999. Patients' sera with incomplete data on stage and sex and duplicate sera were excluded, leaving 300,000 sera for analysis. The seroprevalence for syphilis was 1.77% (n = 5320), as determined by a positive TPHA test. It was significantly higher in male than in female patients (2.03% versus 1.58%, P<0.001). Of the patients reactive in the TPHA test, 3257 (61.2%) were negative in the VDRL. With regard to reactivity in VDRL testing, 2799 patients (0.92%) of the study population were positive, of whom 736 (26%) were biological false positive. BFP reactivity was found in 0.24% of all patients and was significantly higher in women than in men (0.27% versus 0.20%, P<0.001) and in patients over 60 years of age (0.34%) as compared with those under 60 (0.25%, P<0.001). This proportion might be even higher, as reactivity in the VDRL at 1:0 and 1:2 dilutions without a positive treponemal test was not reported. The subgroup of HIV-positive patients (n = 1415) revealed a 10-fold higher rate of BFP tests (2.1% versus 0.24), an effect being statistically significant. In a low syphilis prevalence population, BFP reactions comprise a high proportion of all VDRL reactors. Therefore, the use of the VDRL as a screening procedure is challenged.