Summary. Hepatitis C virus (HCV) infection results in several changes in mitochondrial function including increased reactive oxygen species (ROS) production and greater sensitivity to oxidant, Ca2+ and cytokine-induced cell death. Prior studies in protein over-expression systems have shown that this effect can be induced by the core protein, but other viral proteins and replication events may contribute as well. To evaluate the specific role of core protein in the context of viral replication and infection, we compared mitochondrial sensitivity in Huh7-derived HCV replicon bearing cells with or without core protein expression with that of cells infected with the JFH1 virus strain. JFH1 infection increased hydrogen peroxide production and sensitized cells to oxidant-induced loss of mitochondrial membrane potential and cell death. An identical phenomenon occurred in genome-length replicons-bearing cells but not in cells bearing the subgenomic replicons lacking core protein. Both cell death and mitochondrial depolarization were Ca2+ dependent and could be prevented by Ca2+ chelation. The difference in the mitochondrial response of the two replicon systems could be demonstrated even in isolated mitochondria derived from the two cell lines with the ‘genome-length’ mitochondria displaying greater sensitivity to Ca2+-induced cytochrome c release. In vitro incubation of ‘subgenomic’ mitochondria with core protein increased oxidant sensitivity to a level similar to that of mitochondria derived from cells bearing genome-length replicons. These results indicate that increased mitochondrial ROS production and a reduced threshold for Ca2+ and ROS-induced permeability transition is a characteristic of HCV infection. This phenomenon is a direct consequence of core protein interactions with mitochondria and is present whenever core is expressed, either in infection, full-length replicon-bearing cells, or in over-expression systems.