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OBJECTIVES To estimate the prevalence, incidence and determinants of herpes simplex type 2 (HSV-2) infection, and associations between HSV-2 and incident HIV infection, among women at higher risk for HIV infection in Beira, Mozambique. METHODS Between 2009 and 2012, 411 women aged 18-35 years at higher risk of HIV acquisition (defined as having had two or more sexual partners in the month prior to study enrollment) were enrolled and followed monthly for one year. At each study visit, they were counseled, interviewed, and tested for HSV-2 and HIV antibodies. RESULTS The HSV-2 prevalence at baseline was 60.6% (95% CI: 55.7% -65.4%). Increasing age (aOR = 2.94, 95% CI: 1.74-4.97, P<0.001 and aOR = 3.39, 95% CI: 1.58-7.29, P = 0.002 for age groups of 21-24 and 25-35 years old respectively), lower educational level (aOR = 1.81, 95% CI: 1.09-3.02, P = 0.022), working full time (aOR = 8.56, 95% CI: 1.01-72.53, P = 0.049) and having practiced oral sex (aOR = 3.02, 95% CI: 1.16-7.89, P = 0.024) were strongly associated with prevalent HSV-2 infection. Thirty one participants seroconverted for HSV-2 (20.5%; 95% CI: 14.4% -27.9%) and 22 for HIV during the study period. The frequency of vaginal sex with a casual partner using a condom in the last 7 days was independently associated with incident HSV-2 infection (aOR = 1.91, 95% CI: 1.05-3.47, P = 0.034). Positive HSV-2 serology at baseline was not significantly associated with risk of subsequent HIV seroconversion. CONCLUSIONS Young women engaging in risky sexual behaviors in Beira had high prevalence and incidence of HSV-2 infection. Improved primary HSV-2 control strategies are urgently needed in Beira.