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Dramatic increases have occurred in sexually transmitted diseases (STD) and in sexual risk behaviour among homosexual men in Amsterdam and internationally. We investigated whether these trends indicate a resurgence of the HIV epidemic.HIV incidence was determined among homosexual attendees of an STD clinic in Amsterdam, who had participated in semi-annual anonymous unlinked cross-sectional HIV prevalence studies from 1991 to 2001. Stored HIV-seropositive samples were tested with a less-sensitive HIV assay and, if non-reactive, were further tested for the presence of antiretroviral drugs, indicative of the use of highly active antiretroviral therapy. Seropositive men who tested non-reactive on the less-sensitive assay and had not used antiretroviral drugs were classified as recently infected (< 170 days). Annual HIV incidence and its changes were examined.Among 3090 homosexual participants (median age 34 years), 454 were HIV infected, of whom 37 were recently infectioned. From 1991 to 2001 the overall incidence was 3.0 infections/100 person-years. Incidence increased over time (P = 0.02) and, strikingly, the increase was evident in older (≥ 34 years) men (P < 0.01), but not in the young. Of men recently infected, 84% (n = 31) were unaware of their infection and 70.3% (n = 26) had a concurrent STD. These 26 men reportedly had sex with a total of 315 men in the preceding 6 months.HIV incidence is increasing among homosexual attendees of an STD clinic. It is imperative to trace recently infected individuals, because they are highly infectious, and can thus play a key role in the spread of HIV.