Development of serological and nucleic acid testing (NAT) has revolutionized hepatitis C virus (HCV) diagnosis. Although third generation anti-HCV enzyme immunoassays (EIAs) are very effective for testing high prevalence populations, confirmatory testing is still necessary when these tests are applied to populations with a low HCV prevalence to exclude false positive results. Limitations of third generation anti-HCV EIAs include: the relatively prolonged time between acute infection and detection of seroconversion (which typically requires at least 5-6 weeks); delayed seroconversions in immunocompromised hosts (requiring months to years); and the inability of serological tests to confirm active HCV infection. In contrast, nucleic acid testing (NAT) can directly detect HCV RNA in serum, plasma or tissue and thereby confirm active infection as well as narrow the window between infection and HCV detection to as little as 1-2 weeks. Commercial NAT assays are now highly sensitive, specific, and reproducible and have largely replaced unreliable home brew nucleic acid amplification assays. Qualitative commercial NAT are typically more sensitive than quantitative assays and therefore the method of choice to confirm active infection. Given the efficacy of combination therapy with interferon/ribavirin and newer antiviral agents under development, HCV infection may become curable, which will likely impact future disease transmission. As the therapeutic costs are currently very high, there is clearly a need to assess the utility of quantitative NAT and to further evaluate the role of HCV genotyping to optimize antiviral therapy. Thus for the foreseeable future, a combination of both serological tests and NAT will be required for cost-effective HCV diagnosis and monitoring.