This study estimates HIV incidence in the United States using laboratory technologies that differentiate recent from long-standing infections. Data from 22 states with HIV incidence surveillance were analyzed using the BED HIV-1 capture enzyme immunoassay to classify infections as recent or long-standing. The estimated number of new HIV infections in the U.S. in 2006 was 56,300 (95% CI 48,200–64,500), with an incidence rate of 22.8 per 100,000 population (95% CI 19.5–26.1). Forty-five percent of infections were among blacks, and 53% among men who have sex with men (MSM). Back-calculation of HIV incidence from 1977–2006 using HIV diagnoses and AIDS incidence data also estimated 55,400 new infections per year for 2003–2006, indicating an increase in HIV incidence in the mid-1990s, followed by a slight decline after 1999 and stability thereafter. The study used a stratified extrapolation approach, adjusting for reporting delays and risk factor redistribution. It also employed an extended back-calculation method using HIV diagnoses and AIDS incidence data to estimate annual HIV incidence. The results showed that HIV incidence rose sharply after 1977, peaked in 1984–85, then declined, with a resurgence in the late 1990s. Incidence among MSM remained high, while incidence among blacks was significantly higher than among whites. The study highlights the disproportionate distribution of HIV infection among blacks and Hispanics compared to whites. The study's findings suggest that HIV incidence in the U.S. remains concentrated among MSM and African Americans. The estimates are the first direct measurement of HIV incidence using laboratory technologies previously used in clinic-based settings. The study underscores the importance of accurate HIV incidence data for planning and evaluating prevention efforts and resource allocation. The results also indicate that without direct incidence data, delays may occur in recognizing resurgences of HIV infections among certain populations, potentially delaying necessary prevention efforts. The study's methods and results provide a more accurate picture of the HIV epidemic and support improved prevention strategies.This study estimates HIV incidence in the United States using laboratory technologies that differentiate recent from long-standing infections. Data from 22 states with HIV incidence surveillance were analyzed using the BED HIV-1 capture enzyme immunoassay to classify infections as recent or long-standing. The estimated number of new HIV infections in the U.S. in 2006 was 56,300 (95% CI 48,200–64,500), with an incidence rate of 22.8 per 100,000 population (95% CI 19.5–26.1). Forty-five percent of infections were among blacks, and 53% among men who have sex with men (MSM). Back-calculation of HIV incidence from 1977–2006 using HIV diagnoses and AIDS incidence data also estimated 55,400 new infections per year for 2003–2006, indicating an increase in HIV incidence in the mid-1990s, followed by a slight decline after 1999 and stability thereafter. The study used a stratified extrapolation approach, adjusting for reporting delays and risk factor redistribution. It also employed an extended back-calculation method using HIV diagnoses and AIDS incidence data to estimate annual HIV incidence. The results showed that HIV incidence rose sharply after 1977, peaked in 1984–85, then declined, with a resurgence in the late 1990s. Incidence among MSM remained high, while incidence among blacks was significantly higher than among whites. The study highlights the disproportionate distribution of HIV infection among blacks and Hispanics compared to whites. The study's findings suggest that HIV incidence in the U.S. remains concentrated among MSM and African Americans. The estimates are the first direct measurement of HIV incidence using laboratory technologies previously used in clinic-based settings. The study underscores the importance of accurate HIV incidence data for planning and evaluating prevention efforts and resource allocation. The results also indicate that without direct incidence data, delays may occur in recognizing resurgences of HIV infections among certain populations, potentially delaying necessary prevention efforts. The study's methods and results provide a more accurate picture of the HIV epidemic and support improved prevention strategies.