The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV-positive and -negative individuals and cultured for up to 40 days. Biliary cells from HCV-negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV-negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV-RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single-strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.