HPTN 064, or ISIS (The Women’s HIV Seroincidence Study), was designed to estimate the overall new HIV infection rate in women at risk for HIV in the US. A total of 2,099 women between the ages of 18 and 44 years were enrolled from May 2009 to July 2010. Women were enrolled from 10 communities in six distinct geographical areas in the northeast and southeast regions of the US; Atlanta, GA, Raleigh-Durham, NC, Washington D.C., Baltimore, MD, Newark, NJ, New York City, NY. Eighty-eight percent of the participants in the study were black, 12% identified as Hispanic/Latina. The study used a novel approach to recruit participants, focusing on geographic areas of the US with the highest HIV prevalence rates in women. Women without a prior positive HIV test living in areas with high HIV prevalence and poverty were eligible for enrollment and were interviewed about many key aspects of their lives including mental health, sexual behavior, history of sexually transmitted infections (STIs), domestic violence, social support, financial insecurity and health care utilization. Study results indicated that the HIV incidence rate for U.S. women living in areas hardest hit by the epidemic was much higher than the overall estimated incidence rate in the U.S. for black adolescent and adult women. HPTN 064 found an HIV incidence of 0.24% in the study cohort of 2,099 women (88% black), a rate that was fivefold higher than that estimated for black women overall by the Centers for Disease Control and Prevention (CDC). The rate noted in HPTN 064 was comparable to estimated HIV incidence rates in the general population in several countries in sub-Saharan Africa including the Congo (0.28 %) and Kenya (0.53%), underscoring the substantial ongoing HIV transmission within specific US populations, including women at risk as defined in this study.