This venue-based, cross-sectional study reports on human immunodeficiency virus (HIV) prevalence and behaviour of 649 men who have sex with men (MSM) in Antwerp and Ghent, Flanders, Belgium, from October 2009 to March 2010. Using time-location sampling, we found that HIV prevalence in MSM who attended different types of venue ranged from a high of 14.5% (95% CI: 8.9–20.1; n=22 in cruising venues to 4.9% (95% CI: 1.9–7.9; n=10) in more general gay venues to 1.4% (95% CI: 0.0–3.6; n=3) at younger MSM venues. Of those who tested HIV positive (n=35, five were unaware of their HIV status or self-reported as being HIV negative. One in five respondents were of non-Belgian nationality. The results showed relatively high rates of testing for HIV (52.2%; 95 % CI: 47.8–56.2; n=288) and other sexually transmitted infections (STIs) (57.4%; 95% CI: 52.6–62.0; n=248) in the last 12 months. A majority of the men (n=233) used condoms consistently during their last anal sexual contact with a casual partner; however, HIV-positive men who were aware of their serostatus (n=30) reported less condom use with casual partners. This is the first such study in Belgium and the results constitute the evidence base for local, targeted interventions. Furthermore, our findings underscore the need for European cross-border cooperation to prevent HIV infection and other STIs among MSM.