Drug-resistant minority viral variants can pre-exist in the viral quasispecies of chronically infected hepatitis C virus (HCV) patients and can emerge gradually upon drug treatment. When heterogeneous clinical samples are tested for drug susceptibility in a chimeric replicon-based phenotyping assay, biphasic dose-response curves may be observed. The effect of drug-resistant minority viral variants on the biphasic phenotype of mixtures was assessed in detail.Susceptibility of mutant/wild-type mixtures containing minorities of NS3 mutants with different replication capacities and susceptibilities to protease inhibitors were tested in a transient replicon assay. The contribution of both variants in the mixture to the overall replication level was described with an E(max) model.The 90% and 99% effective concentrations (EC(90) and EC(99), respectively) provide a more accurate measure of the susceptibility of the population than the determination of EC(50) values. Reduced susceptibility at the EC(50) level correlated with the replication capacity of the NS3 mutant in the mixture. Using replication-enhanced mutant/wild-type mixtures demonstrated that the relative difference between the replication capacity of the variants present in the mixture results in biphasic dose-response curves. Modelling revealed that in mixtures containing wild-type and resistant variants with low replication capacity, the contributions of the wild-type variants are higher than expected from the replication level of the replicons transfected alone.Differences in the replication capacity of variants present in HCV replicon-based phenotype assays can lead to biphasic dose-response curves. Using EC(90) or EC(99) values increases the sensitivity of the assay to minor variants.