To improve the long-term efficacy of interferon (IFN) for treatment of chronic hepatitis C virus (HCV) infection, we proposed induction therapy with twice-a-day IFN-beta injection, This study was intended to clarify the antiviral mechanism. Thirty patients were randomly assigned to two groups: group A (twice-a-day therapy) received 3 MU IFN-beta intravenously (i.v.) twice a day for 2 weeks; group B (once-a-day therapy) received 6 MU of IFN-beta daily, HCV RNA, IFN-beta, alanine aminotransferase (ALT), 2'5'-oligoadenylate synthetase (2'5'-AS) activity, and beta(2)-microglobulin in serum were compared between the two groups during the first 2 weeks of IFN therapy. The clearance rate of serum HCV RNA in group A (86.7%) was significantly higher than that in group B (13.3 %) at day 3 (p = 0.0006), No accumulation of IFN-beta was shown in serum throughout the therapy, The ratio (day 3/day 1) of 2'5'-AS activity was significantly higher in group A. Multivariate analysis indicated twice-a-day IFN-beta injection therapy led to significantly early clearance of circulating HCV. Twice-a-day IFN-beta injection therapy could induce biologically enhanced antiviral activities and be an efficient induction therapy for eradication of HCV.