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To estimate HIV-1 prevalence among women attending an antenatal clinic in the Gulu District (North Uganda) and, based on these data, among the district's female population.Anonymous HIV-1 screening was performed for 8555 antenatal clinic attendees aged 15–39 years in the period 1993–1997. The results were used to estimate the prevalence among the district's female population, accounting for differences in fertility rates by HIV-1 serostatus.Among antenatal clinic attendees, HIV-1 prevalence showed a significant linear decrease (P < 0.001), from 26.0% in 1993 [95% confidence Interval (CI), 23.2–29.0%] to 16.1% in 1997 (95% CI, 14.8–17.5%). This decrease was mostly due to a marked decrease until 1995 (14.3%; 95% CI, 12.7–16.0%) and was more pronounced among women aged under 30 years (P < 0.001), from both urban and rural areas (P < 0.001). The risk of being infected was higher among women from urban areas (Gulu Municipality), both over the entire period (adjusted prevalence proportion ratio = 1.54; 95% CI, 1.40–1.68) and by individual year. The estimated prevalence for the 15–39-year-old female population, standardized by age and area of residence, decreased from 25.4% in 1993–1994 to 17.8% in 1996–1997; these rates were 1.22 and 1.28 times higher, respectively, than those among antenatal clinic attendees.The trend of decrease among young women, for whom changes in HIV-1 prevalence more closely reflect incidence, could be partially due to a reduction in risk behaviour and a consequent decreasing incidence. Differences in fertility rates by HIV-1 serostatus should be addressed when using antenatal clinic data to estimate prevalence among the general female population.