This study aims at estimating the recent trends in HIV-1 prevalence and the factors associated with infection among pregnant women in the Gulu District of north Uganda, a rural area severely affected by civil strife. In 2000-2003, a total of 4459 antenatal clinic attendees of Lacor Hospital were anonymously tested for HIV-1 infection. The overall and age-specific prevalence did not show any significant trend over time. The age-standardized prevalence slightly declined, from 12.1% in 2000 to 11.3% in 2003. Increased age [20-24 years: adjusted odds ratio (AOR) 1.63; 95% CI 1.18-2.25; >or=25 years: AOR 2.56; 95% CI 1.91-3.44], residence in urban areas (AOR 1.76; 95% CI 1.41-2.18), being unmarried (AOR 1.60; 95% CI 1.27-2.01), increased age of partner (25-34 years: AOR 1.87; 95% CI 1.29-2.73; >or=35 years: AOR 2.68; 95% CI 1.72-4.16), modern occupation of partner (AOR 1.98; 95% CI 1.53-2.58), and short time of residence at the current address (AOR 1.36; 95% CI 1.05-1.76) were associated with infection. The HIV-1 prevalence in this rural district is high and similar to that observed in urban antenatal clinics, probably reflecting the effect of the last 18 years of civil strife.