The Procleix HIV-1/HCV Assay is a high-throughput nucleic acid test for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA during blood donor screening. This study evaluated the clinical sensitivity of the Procleix assay and assessed the assay's ability to identify HIV-1- and HCV-infected individuals undetected by standard serologic tests. Plasma samples were obtained prospectively from 539 individuals at high risk for HIV-1 and HCV infection at seven clinics affiliated with Johns Hopkins University. Samples were tested in the Procleix HIV-1/HCV Assay and, if reactive, were then tested in the Procleix HIV-1 and HCV discriminatory assays to differentiate the source of viral RNA positivity. Of these 539 subjects, 287 (53.2%) tested reactive in the Procleix HIV-1/HCV Assay. In discriminatory assay testing, 12 of 287 subjects (4.2%) were reactive for HIV-1 RNA only, 260 (90.6%) were reactive for HCV RNA only, and 11 (3.8%) were coinfected with HIV-1 and HCV. The clinical sensitivity for samples tested neat was 100% for HIV-1 and 99.3% for HCV. Three subjects with Procleix HCV reactive/seronegative results seroconverted upon follow-up and were confirmed as Procleix HCV yield cases. The Procleix HIV-1/HCV Assay is a highly sensitive test that detects ongoing and early HIV-1 and HCV infection in a significant number of subjects at high risk for these diseases. Confirmation of Procleix yield cases upon follow-up demonstrated the ability of the Procleix HIV-1/HCV Assay to detect the presence of HIV-1 and HCV in blood earlier than standard serologic tests.