Background: Screening large numbers of persons in a population with low prevalence of a disease leads to many false-positives. However, populations with low HCV prevalence may sometimes be recommended for HCV screening, for instance patients or healthcare workers after a possible healthcare-related exposure. Objectives: We determined the percentage of true vs false-positive HCV antibody (anti-HCV) test results among 2007-2012 participants in the National Health and Nutrition Examination Study (NHANES), a nationally representative study with approximately 1% HCV infection prevalence, much lower than in groups typically recommended for HCV screening. Study design: Anti-HCV test confirmation was performed using a recombinant immunoblot assay (RIBA) test and follow-up HCV RNA testing. Results: Overall, of 22,359 NHANES participants tested, 479 (2%) were anti-HCV screening reactive and 477 were tested for RIBA; of these 323 (68%) confirmed as true positive and 105 (22%) were false-positives. Many others (49, 10%) were RIBA indeterminate and likely false-positive. Because of these false positive tests, the overall prevalence of chronic infection among those testing anti-HCV screening reactive was much lower (218, 51%) than would be expected due to disease clearance alone (approximately 80%). Conclusions: All screening anti-HCV positive tests should be followed by an HCV RNA test, in order to confirm whether the patient has current infection so that infected persons can be referred to care and treatment to avoid the significant morbidity and mortality associated with chronic HCV infection. Published by Elsevier B.V.