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To determine (trends in) HIV incidence among MSM\ who have recently had postexposure prophylaxis (PEP) prescribed in Amsterdam, compared with MSM participating in the Amsterdam Cohort Studies (ACS).We used data from MSM who were prescribed PEP in Amsterdam between 2000 and 2009, who were HIV-negative at the time of PEP prescription and had follow-up HIV testing 3 and/or 6 months after PEP prescription (n = 395). For comparison, cohort data from MSM participating in the ACS in the same period were used (n = 782). Poisson log-linear regression analyses were performed to model trends in HIV incidence and identify differences in HIV incidence between both cohorts at different time points.Between 2000 and 2009, among MSM who were prescribed PEP, an overall HIV incidence of 6.4 [95% confidence interval (CI) 3.4–11.2] per 100 person-years was found, compared with an HIV incidence of 1.6 (95% CI 1.3–2.1) per 100 person-years among MSM participating in the ACS (P < 0.01). In both cohorts, an increasing trend in HIV incidence over time was observed [incidence rate ratio (IRRpercalendaryear) 1.3 (95% CI 0.9–1.7) and 1.1 (95% CI 1.0–1.2) among MSM prescribed PEP and MSM of the ACS, respectively]. The difference in HIV incidence between both cohorts was most evident in more recent years [IRRPEPversusACS in 2009 4.8 (95% CI 2.0–11.5)].Particularly in more recent years, MSM recently prescribed PEP had a higher HIV incidence compared with MSM participating in the ACS, indicating ongoing sexual risk behaviour.