Investigation of the hepatitis C virus (HCV) life cycle and the evaluation of novel antiviral strategies are limited by the lack of an efficient cell culture system. Therefore, continuous human cell lines inducibly expressing the entire HCV open reading frame were generated with use of a tetracycline-regulated gene expression system. HCV transgenes were found to be chromosomally integrated in a head-to-tail configuration. Northern blot analyses revealed a tightly regulated unspliced transcript of approximately 9 kilobases (kb). HCV structural and nonstructural proteins were faithfully processed, indicating that the cellular and viral proteolytic machineries and posttranslational modification pathways are fully functional in these cell lines. Steady state expression levels could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Kinetic analyses revealed a half-life of less than 1 hour for the HCV RNA whereas a half-life of approximately 9.5, 12, 11, and 10 hours was found for core, NS3, NS4A, and NS5A proteins, respectively. Viral proteins were found to colocalize in the cytoplasm in a pattern characteristic of the endoplasmic reticulum. High-level expression of HCV proteins in the fully induced state was toxic to the cells. These cell lines provide a unique in vitro system to analyze structural and functional properties of HCV proteins, their interactions with cellular proteins and pathways, and the requirements for HCV morphogenesis. In addition, they should prove useful for the evaluation of novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context.