We conducted a randomized controlled trial to assess the efficacy of twice-a-day administration of natural interferon beta (IFNbeta) as an induction of IFN therapy for chronic hepatitis C. Seventy-one patients with chronic hepatitis C were enrolled into the trial and randomly assigned into three treatment groups. Six million units (MU) of IFNbeta were administered once-a-day for the first 4 weeks, and then thrice weekly for 12 weeks in 20 patients (once-a-day group). Three milion units of IFNbeta were administered twice-a-day for the first 2 weeks, 6 MU once-a-day for the next 2 weeks, and then thrice weekly for 12 weeks in 23 patients (twice-a-day+beta group), or 6 MU of lymphoblastoid IFNalpha were administered thrice weekly for the last 12 weeks instead of IFNbeta in 28 patients (twice-a-day+alpha group). Four patients in once-a-day group (20%), 9 in twice-a-day+beta group (39%), and 12 in twice-a-day+alpha group (43%) obtained sustained response. Sustained response rate in twice-a-day groups was higher than in once-a-day group, although there was no statistical significance. The present study suggested the possible superiority of twice-a-day administration of IFNbeta as an induction therapy to once-a-day administration, but further studies are needed to confirm this regimen.