Objectives HIV diagnosis rates in men who have sex with men (MSM) began increasing in Australia 10 years ago, and there has been a major resurgence of syphilis. We determined predictors of HIV positivity and seroconversion among MSM in Victoria, Australia. Methods: We conducted a retrospective longitudinal analysis of data from MSM who underwent HIV testing between April 2006 and June 2009 at three primary care clinics. Logistic regression was used to determine predictors of HIV positivity and seroconversion. Results: During the study period, 7857 MSM tested for HIV. Overall HIV positivity was 1.86% (95% confidence interval (CI): 1.6-2.2). There were 3272 repeat testers followed for 4837 person-years (PY); 60 seroconverted and HIV incidence was 1.24 (95% CI: 0.96-1.60) per 100 PY. Independent predictors of HIV seroconversion were: an infectious syphilis diagnosis within the last 2 years (adjusted hazard ratio (AHR)=2.5, 95% CI: 1.1-5.7), reporting six or more anal sex partners in the past 6 months (AHR=3.3, 95% CI: 1.8-6.3), reporting an HIV-positive current regular partner (AHR=3.4, 95% CI: 1.1-10.6) and reporting inconsistent condom use with casual partners in the past 6 months (AHR=4.4, 95% CI: 1.7-11.5). Conclusion: Our results call for HIV prevention to target high-risk MSM, including men with a recent syphilis diagnosis or a high numbers of partners, men who have unprotected anal sex with casual partners and men in serodiscordant relationships. The HIV incidence estimate will provide a baseline to enable public health officials to measure the effectiveness of future strategies.