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We explored seroguessing (serosorting based on the assumption of HIV seroconcordance) and casual unprotected anal intercourse (UAIC) associated with seroguessing. The ongoing Positive Health and Health in Men cohorts, Australia, provided data for trends in seroconcordant UAIC and HIV disclosure to sex partners. In event-level analyses, we used log-binomial regression adjusted for within-individual correlation and estimated prevalence rate ratios (PRRs) and 95% confidence intervals (95% CIs) for the association between the knowledge of a casual partner`s seroconcordance and UAIC. UAIC and HIV disclosure significantly increased during 2001-2006. HIV-positive men knew partners were seroconcordant in 54% and assumed it in 13% of sex encounters (42 and 17% among HIV-negative men). Among HIV-positive men, the likelihood of UAIC was higher when a partner`s status was known (Adjusted PRR = 5.17, 95% CI: 3.82-7.01) and assumed seroconcordant because of seroguessing (Adjusted PRR = 3.70, 95% CI: 2.56-5.35) compared with unknown. Among HIV-negative men, the likelihood of UAIC was also higher when a partner`s status was known (Adjusted PRR = 1.88, 95% CI: 1.58-2.24) and assumed seroconcordant (Adjusted PRR = 2.12, 95% CI: 1.72-2.62) compared with unknown. As levels of UAIC remain high, seroguessing increasingly exposes gay men to the risk of HIV infection. Because both HIV-positive and HIV-negative men often seroguess, education and prevention programs should address the fact that HIV-negative men who engage in UAI due to this practice may be at high risk of HIV infection. HIV prevention should take into account these contemporary changes in behaviors, especially among HIV-negative gay men.