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In 2008, the first clinic for women involved in high-risk sexual behavior was established in Kampala, offering targeted HIV prevention. This article describes rates, determinants, and trends of HIV incidence over 3 years.A total of 1027 women at high risk were enrolled into a closed cohort. At 3-monthly visits, data were collected on sociodemographic variables and risk behavior; biological samples were tested for HIV and other reproductive tract infections/sexually transmitted infections (RTI/STIs). Hazard ratios for HIV incidence were estimated using Cox proportional hazards regression among the 646 women HIV negative at enrolment.HIV incidence was 3.66/100 person-years (pyr) and declined from 6.80/100 pyr in the first calendar year to 2.24/100 pyr and 2.53/100 pyr in the following years (P trend = 0.003). Sociodemographic and behavioral factors independently associated with HIV incidence were younger age, younger age at first sex, alcohol use (including frequency of use and binge drinking), number of paying clients in the past month, inconsistent condom use with clients, and not being pregnant. HIV incidence was also independently associated with Mycoplasma genitalium infection at enrolment [adjusted hazard ratio (aHR) = 2.28, 95% confidence interval (CI): 1.15 to 4.52] and with Neisseria gonorrhoeae (aHR = 5.91, 95% CI: 3.04 to 11.49) and Trichomonas vaginalis infections at the most recent visit (aHR = 2.72, 95% CI: 1.27 to 5.84). The population attributable fractions of HIV incidence for alcohol use was 63.5% (95% CI: 6.5 to 85.8) and for treatable RTI/STIs was 70.0% (95% CI: 18.8 to 87.5).Alcohol use and STIs remain important risk factors for HIV acquisition, which call for more intensive control measures in women at high risk. Further longitudinal studies are needed to confirm the association between M. genitalium and HIV acquisition.