BACKGROUND: We investigated the prevalence and protective value of serosorting [ie, establishing HIV concordance in advance to practice unprotected anal intercourse (UAI)] with casual partners (CP) among HIV-negative men who have sex with men (MSM) using longitudinal data from 2007 to 2011. METHODS: Men of the Amsterdam Cohort Studies were tested biannually for HIV-1 antibodies and filled in questionnaires about sexual behavior in the preceding 6 months. HIV incidence was examined among men who practiced UAI, UAI with serosorting, or consistent condom use, using Poisson regression. RESULTS: Of 445 MSM with CPs, 31 seroconverted for HIV during a total follow-up of 1107 person-years. Overall observed HIV incidence rate was 2.8/100 person-years. Consistent condom use was reported in 64%, UAI in 25%, and UAI with serosorting in 11% of the 2137 follow-up visits. MSM who practiced serosorting were less likely to seroconvert [adjusted incidence rate ratio (aIRR) = 0.46; 95% confidence interval (CI): 0.13 to 1.59] than MSM who had UAI, but more likely to seroconvert than MSM who consistently used condoms (aIRR = 1.32; 95% CI: 0.37 to 4.62), although differences in both directions were not statistically significant. MSM who consistently used condoms were less likely to seroconvert than MSM who had UAI (aIRR = 0.37; 95% CI: 0.18 to 0.77). DISCUSSION: The protective effect for serosorting we found was not statistically significant. Consistent condom use was found to be most protective against HIV infection. Larger studies are needed to demonstrate whether serosorting with CPs offers sufficient protection against HIV infection, and if not, why it fails to do so.