We tested serial serum samples for hepatitis C virus RNA from patients undergoing treatment for chronic hepatitis C with interferon-alpha using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 20 patients with chronic hepatitis who had serum antibody to hepatitis C virus (anti-C100-3). Before therapy, hepatitis C virus RNA was detected in 18 (90%) and 20 (100%) patients using primer sets derived from the NS3 region or the 5'-noncoding region of hepatitis C virus, respectively. Hepatitis C virus RNA became undetectable in all patients whose ALT level fell into the normal range during therapy. However, hepatitis C virus RNA reappeared in all patients whose ALT levels rose again after therapy, usually before the relapse. In patients whose ALT levels did not become normal, hepatitis C virus RNA did not disappear during therapy. Thus therapy with interferon-alpha appears to be beneficial in chronic hepatitis C because of its suppressive effects on hepatitis C virus replication. Detection of hepatitis C virus RNA in serum is useful for evaluating the antiviral effect of interferon.