Serum, liver tissue, and peripheral blood mononuclear cells (PBMC) were obtained from 72 patients with chronic hepatitis C, 19 of whom had received interferon-alpha (IFN-alpha) treatment. Hepatitis C virus (HCV) RNA was detected by polymerase chain reaction in the serum and liver of all patients with relapse after treatment and in 51 of 53 (96%) sera, 28 of 29 (97%) livers, and all of 13 (100%) PBMC from untreated patients but not in 3 patients with completely sustained responses. Minus-stranded HCV RNA was found in 5 of 14 (35%) sera, 16 of 17 (94%) livers, and 7 of 13 (53%) PBMC from untreated patients. The results indicate that HCV RNA is present not only in serum and liver but also in PBMC from the vast majority of untreated anti-HCV-seropositive patients and also in patients who respond to IFN-alpha treatment but then relapse. HCV replicates in the liver of almost all patients, but replication may possibly also occur in PBMC. By contrast, HCV RNA seems to be eradicated in patients with completely sustained responses.