BACKGROUND: Concentration of viral particles by ultracentrifugation of serum prior to PCR allows detection of hepatitis C virus (HCV) RNA in patients with undetectable viral RNA by conventional PCR assays. AIM: To analyse if HCV-RNA is detected after serum ultracentrifugation in chronic hepatitis C patients with a sustained virological response to antiviral therapy (defined as serum HCV-RNA negativity by conventional assays 6 months after the end of therapy). METHODS: HCV-RNA was tested using real-time PCR in ultracentrifuged sera collected during the post-treatment follow-up (mean: 42 +/- 27 months) in 57 sustained virological responders (SVR). RESULTS: After serum ultracentrifugation, HCV-RNA was detected on at least one occasion during the follow-up in 29/57 (51%) SVR. Thirteen (23%) of these 57 SVR suffered a reactivation 18 +/- 8 months after the end of therapy (reappearance of serum HCV-RNA detectable by conventional assays). Among reactivated patients, 11/13 (85%) had HCV-RNA in ultracentrifuged serum samples (detectable 10 +/- 5 months before reactivation), while HCV-RNA was positive after ultracentrifugation in 18/44 (41%) long-term SVR (P = 0.01). Persistence of detectable HCV-RNA after serum ultracentrifugation was associated with reactivation (P = 0.001). CONCLUSIONS: Serum ultracentrifugation prior to PCR allows detection of HCV-RNA in SVR and its persistence may predict late reactivation.