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To revise the national HIV estimates and quantify the magnitude of the HIV epidemic in Ukraine at the end of 2007.Internationally recommended methods—the Workbook and Spectrum—were employed to generate the estimates. This enables comparison of results with other countries using the same methodology.Estimation of the size of most at-risk populations nationally was performed using capture-recapture, multiplier and triangulation methods. HIV prevalence among most at-risk populations was estimated by linked HIV sentinel and behavioural surveillance among injecting drug users, and men who have sex with men, and unlinked sentinel surveillance among sex workers. The range of HIV prevalence and extrapolation for populations at lower risk were determined by consensus among national stakeholders. Results were reviewed by national stakeholders and endorsed by the government of Ukraine.At the end of 2007, an estimated 395 000 adults (range 230 000–573 000) aged 15–49 were living with HIV in Ukraine. Adult HIV prevalence was estimated at 1.63%, which represents the highest adult HIV prevalence of any country in Europe.The HIV epidemic in Ukraine continues to grow at a record pace, concentrated among most at-risk populations, the majority of whom are unaware of their HIV status. The results emphasise the need to accelerate the coverage and quality of prevention programmes among most at-risk populations and their sexual partners.