Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-alpha), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-alpha concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-alpha treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-alpha treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began approximately 12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-alpha-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-alpha inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-alpha. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-alpha-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-alpha suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-alpha inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-alpha may be inhibiting HCV in vivo.