Thirty years of civil war in the Sudan have resulted in the isolation of the southern provinces which border Central and East Africa. Consequently, little is known about the epidemiology of HIV-1 infection in this region. To estimate the prevalence of HIV-1 infection in southern Sudan and the risk factors associated with disease transmission, a seroepidemiologic survey was conducted in the township of Juba. Study subjects invited to participate in this study included medical outpatients, inpatients hospitalized for active tuberculosis, and female prostitutes. A total of 401 subjects participated in the study. HIV-1 infection was confirmed in 25 subjects. The prevalence of HIV-1 infection was 19% (8/42) among tuberculosis patients, 16% (8150) among prostitutes, and 3% (9/309) among outpatients. A significantly higher prevalence of HIV-1 infection was found among female prostitutes when compared to female outpatients: 16% (8150) vs. 2% (4/178), P < 0.001. Correspondingly, the prevalence of seropositives was significantly higher among male outpatients reporting a history of sexual relations with prostitutes during the prior 10 years compared to male outpatients denying relations with prostitutes: 14% (5/37) vs. 0% (0/ 941, P = 0.001. A history of a sexually transmitted disease (STD) was also associated with HIV-1 infection among male outpatients. The findings of this study indicate that HIV-1 infection is highly prevalent in southern Sudan and that prostitutes and their sexual partners represent a major reservoir of HIV infection in this population. This epidemiologic pattern resembles that seen in the African nations neighboring southern Sudan. Further spread of HIV-1 to northern Sudan could occur because of the movement of military personnel and refugees between the North and South of Sudan.