To improve the efficacy of interferon (IFN) therapy for chronic hepatitis C, we proposed a therapy with twice-a-day injection of IFN beta as the induction. To assess its biological enhancement, we compared antiviral activities in vitro using intermittent treatment schedules simulating the clinical condition. FL cells were treated with IFN beta twice in 12 h interval (Single treatment, 1000 and 0 IU/ml; Double treatment, 500 IU/ml each) and challenged with Sindbis virus. Antiviral activities were determined with 50% cytopathic effect. Activities and mRNA expressions of 2'5'oligoadenylate synthetase (2'5'AS) were also examined. Single treatment showed its peak activity at 9 h, while Double treatment was at 3 h after the second treatment. Double treatment had a significantly higher peak activity. The up-regulated activities of 2'5'AS lasted much longer with Double treatment. The present findings demonstrated Double treatment could induce efficient biological enhancement, which is thought based on the priming effect.