Objectives: The number of cases of infectious syphilis is increasing rapidly across England and Wales. Concern has been expressed about diagnostic delay and its potential impact on patient care. A standard operating procedure for the serological diagnosis of syphilis has recently been developed by the Health Protection Agency. This study aimed to audit clinical and laboratory practice in England and Wales against this standard. Methods: All microbiology departments, genitourinary medicine (GUM) clinics and antenatal clinics in England and Wales were invited to complete a web-based questionnaire. Results: The overall response rate was 76%. Practices varied between laboratories. The proportion of microbiology departments performing enzyme immunoassay (EIA), Treponema pallidum particle agglutination assay/T pallidum haemagglutination assay, rapid plasma reagin/Venereal Disease Reference Laboratory and EIA IgM were 94%, 34%, 41% and 10%, respectively. Of these, 57% only perform a single screening assay. The turnaround time for negative results was less than 1 week for 84% of microbiology departments. For positive samples, turnaround times varied from less than 1 week to 6-8 weeks, with 19% of GUM clinics reporting turnaround times of over 3 weeks. Notably, 26% of GUM clinics and 6% of antenatal clinics reported that delays in turnaround time had adversely affected patient management in the past year. Conclusion: This study suggests that there is significant room to improve laboratory turnaround times for the diagnosis of syphilis in England and Wales, and such improvements would be a positive step in limiting the spread of infection and of congenital syphilis.