Background: We have previously reported a procedure for isolating and culturing biliary epithelial cells (BECs). The aim of this study was to reconsider the method for obtaining pure BECs using the mouse gallbladder. Materials and Methods: Cells that were obtained from the gallbladder alone were sorted by fluorescence-activated cell sorting (FACS) for purifying based on the expression of the epithelial cell adhesion molecule (EpCAM). The viability rate was measured based on the negative expression of 7-aminoactinomycin D (7-AAD). Results: More than 75% of cells from the gallbladder were determined to be pure BECs. An analysis of the EpCAM revealed that 73.3% of the cells were 7-AAD-negative. Finally, the 0.82x10(6) pure BECs that survived were obtained and seeded on a collagen gel plate. However, these pure BECs showed almost no proliferation. Conclusion: Pure BECs could be accumulated using FACS. However, the number of BECs was insufficient for the culturing process.