Investigation of the hepatitis C virus (HCV) life cycle is limited by the lack of an efficient cell culture system. Employing a tetracycline-regulated gene expression system we generated a panel of continuous human cell lines allowing the inducible expression and faithful processing of HCV structural proteins as well as of a functional NS2-3 autoprotease. HCV proteins were found in the cytoplasm in a pattern characteristic for the endoplasmic reticulum. High-level expression of HCV proteins was found to be cytotoxic. These cell lines represent a unique in vitro system in which to further investigate the structural proteins of HCV and to evaluate novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context.