After treatment of chronic hepatitis C virus (HCV), infection with interferon-alpha+/-ribavirin in a considerable proportion of patients with a virologic end-of-treatment response (ETR), a relapse during follow-up is observed. The biological background of relapse is unknown. Either antiviral therapy leads to a complete replication arrest and latent virions survive until the end-of-treatment or replication is not completely suppressed. For the latter hypothesis, residual HCV RNA should be detectable dependent on the sensitivity of the HCV RNA assay. In patients from different studies, serum or plasma samples from the end-of-treatment were investigated for the presence of HCV RNA with a standard polymerase chain reaction (PCR)-based assay and retested for comparison with a new highly sensitive transcription-mediated amplification (TMA)-based assay. Generally, in patients with virologic non-response or sustained virologic response (SR), no differences were observed between PCR- and TMA-based assays. In patients with relapse after standard interferon-alpha therapy with or without ribavirin, HCV RNA was detected in 33-36% of end-of-treatment samples previously negative by PCR-based assays. The lower rate of HCV RNA detected by TMA (7%) in end-of-treatment samples from patients after treatment with pegylated interferon-alpha 2a is most likely explained by the longer half-life of pegylated interferon-alpha in comparison with standard interferon-alpha. HCV RNA is detectable at the end-of-treatment by TMA in 7-36% of patients who were HCV RNA negative by reverse transcriptase (RT)-PCR-based methods. These PCR-negative/TMA-positive patients have to be considered as non-responders (NR) with a very low HCV RNA viral load. Future studies will show whether these patients benefit from prolonged antiviral therapy.