Efforts to identify all persons infected with HIV in the United States are driven by the hope that early diagnosis will lower risk behaviors and decrease HIV transmission. Identification of HIV-infected people earlier in the course of their infection with HIV antigen/antibody (Ag/Ab) combination assays (4th-generation HIV assays) should help achieve this goal. We compared HIV RNA nucleic acid test (NAT) results to the results of a 4th-generation Ag/Ab assay (Architect HIV Ag/Ab Combo [HIV Combo] assay; Abbott Diagnostics) in 2,744 HIV antibody-negative samples. Fourteen people with acute HIV infection (HIV antibody negative/NAT positive) were identified; the HIV Combo assay detected nine of these individuals and was falsely negative in the remaining five. All five persons missed by the HIV Combo assay were in the stage of exponential increase in plasma virus associated with acute HIV infection (3, 7, 20, 35, 48). In contrast, most acutely infected persons detected by the HIV Combo assay demonstrated either a plateauing or decreasing plasma viral load. The HIV Combo assay also classified as positive five other samples which were negative by NAT. Taken together, the HIV Combo assay had a sensitivity of 73.7% and a specificity of 99.8%. Using published data, we estimated secondary transmission events had HIV infection in these five individuals remained undiagnosed. Screening of our population with NAT cost more than screening with the HIV Combo assay but achieved new diagnoses that we predict resulted in health care savings that far exceed screening costs. These findings support the use of more sensitive assays, like NAT, in HIV screening of populations with a high prevalence of acute HIV infection.