Debio 025 (D25) is a potent inhibitor of HCV replication both in vitro as in vivo [Hepatology 43:761-70; Hepatology 47:81726]. Here we elaborate on the particular in vitro anti-HCV characteristics of D25. Combining D25 with either interferon-α, ribavirin or STAT-C inhibitors [protease or (non)-nucleoside polymerase HCV inhibitors] resulted in an additive to slightly synergistic antiviral activity in a 3 day antiviral assay. D25 has the unique ability to rapidly clear hepatoma cells from their HCV replicon when used alone or in combination with interferon-α and STAT-C inhibitors. Moreover D25 was able to delay the development of escape variants against several STAT-C inhibitors in colony formation assays. D25 proved as potent against HCV replicons that are resistant to various STAT-C inhibitors as against wild-type HCV. Subgenomic replicons were selected that are resistant to D25 (3 independent selections). Mutations were identified in domain II of the NS5A gene. Reintroduction of these mutations in a WT background partially recovered resistance. D25 resistant (D25r) replicons remained fully susceptible to interferon and several STAT-C inhibitors. Transfection of Huh Lunet cells with D25r replicon RNA, resulted in a partial transfer of the resistance suggesting that also host cell factors are involved in the antiviral activity of D25. D25 forms, given the particular anti-HCV activity and the unique resistance profile, an attractive candidate for the treatment of HCV infections in combination with standard of care and/or STAT-C. Abstract