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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 0.82×106 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.