The Women's Interagency HIV Study comprises the largest U.S. cohort to date of human immunodeficiency virus (HIV)-seropositive women (N = 2,058) with a comparison cohort of seronegative women (N = 568). The methodology, training, and quality assurance activities employed are described. The study population, enrolled between October 1994 and November 1995 through six clinical consortia throughout the United States (totaling 23 sites) represents a typically hard-to-reach study population. More than half of the women in each cohort were living below the federally defined levels of poverty. The women ranged in age from 16 to 73 years; approximately one-quarter self-identified as Latina or Hispanic, over one-half as African-American not of Hispanic origin, and less than 20% as white, non-Hispanic origin. Self-reporting of HIV exposure risk included injection drug use by 34% of the seropositive women and 28% of the seronegative women, heterosexual contact (42% vs 26%), transfusion risk (4% vs 3%) and no identified risk (20% vs 43%). Demographic and HIV exposure risk characteristics of the seropositive cohort were comparable with characteristics of nationally reported AIDS cases in U.S. women. This well characterized cohort of HIV-seropositive and high-risk seronegative women represents a rich opportunity for future studies of HIV disease progression and pathogenesis.