Testing for the presence of antibody to hepatitis C virus (anti-HCV) is recommended for initially identifying persons with hepatitis C virus (HCV) infection (CDC. Recommendations for prevention and control of hepatitis C virus [HCV] infection and HCV-related chronic disease. MMWR 1998;47[No. RR-19] :1-33). Testing for anti-HCV should include use of an antibody screening assay, and for screening test-positive results, a more specific supplemental assay. Verifying the presence of anti-HCV minimizes unnecessary medical visits and psychological harm for persons who test falsely positive by screening assays and ensures that counseling, medical referral, and evaluation are targeted for patients serologically confirmed as having been infected with HCV. However, substantial variation in reflex supplemental testing practices exists among laboratories, and an anti-HCV-positive laboratory report does not uniformly represent a confirmed positive result. These guidelines expand recommendations for anti-HCV testing to include an option for reflex supplemental testing based on screening-test-positive signal-to-cut-off (s/co) ratios. Use of s/co ratios minimizes the amount of supplemental testing that needs to be performed while improving the reliability of reported test results. These guidelines were developed on the basis of available knowledge of CDC staff in consultation with representatives from the Food and Drug Administration and public health, hospital, and independent laboratories. Adoption of these guidelines by all public and private laboratories that perform in vitro diagnostic anti-HCV testing will improve the accuracy and utility of reported anti-HCV test results for counseling and medical evaluation of patients by health-care professionals and for surveillance by public health departments.