OBJECTIVE: Passive surveillance indicates a clear increase in infectious syphilis cases in Victoria, but trends are likely to be influenced by changes in testing. We therefore used testing data from two Melbourne clinics with a high caseload of men who have sex with men to examine infectious syphilis prevalence, time trends and risk factors for infection. METHODS: We extracted laboratory testing data on all males tested for syphilis at the clinics between 2000 and 2004, including demographic information, syphilis test results and laboratory information used to assign HIV status (a HIV positive diagnosis or HIV positive clinical monitoring data). RESULTS: Of 7,906 syphilis tests conducted at the two clinics, overall 1.4% resulted in a diagnosis of infectious syphilis, significantly increasing from 0.5% in 2000 to 2.5% in 2004, p<0.01, and among HIV positive males significantly increasing from 0.0% in 2000 to 6.1% in 2004, p<0.01. The frequency of annual syphilis testing among HIV positive males attending for any STI testing significantly increased from 47.3% in 2000 to 81.4% in 2004, p<0.01. Between 2002 and 2003, the syphilis testing rate per 1,000 consultations increased by 44% (from 65.3 to 94.3 tests per 1,000 consultations) compared with a 500% increase in the infectious syphilis diagnosis rate (from 0.2 to 1.2 positive tests per 1,000 consultations). CONCLUSIONS AND IMPLICATIONS: The increase in infectious syphilis notifications appears unrelated to the observed increased in testing and is more likely to be attributed to increased transmission, highlighting the need for urgent prevention interventions.