Objective To investigate the culture method forepidermal stem cells in vitro. Methods The epidermis was separated from the dermis, and shaken for 10 min in 0.05% trypsin at 37℃ to dissociate into single cells. Epidermal stem cells were selected by rapid attachment to collagen Ⅳ for 10-15 min and cultured on collagen Ⅳ or 3T3 feeder layer. All the cells were grown in DMEM without calcium, supplemented with 10% chelexed fetalbovine serum, 10 μg/L epidermal growth factor, 0.05 mmol/L CaCl2 and 0.8 mg/L hydrocortisone. Cultures were observed for colony formation under a phase constrast microscope. The phenotypes of epidermal stem cells were detected by flow cytometry and immunocytochemistry staining. Results The cells selectedby rapid adherence to collagen Ⅳ formed large colonies at 7～8 days, expressedK19 antigen. The percentages of cells at the G0 and G1 phases of the cell cycle and the percentage of α6briCD71dim cells in the experimental groups were higher than those in the control group. It indiciated that there was a significant difference between the experimental groups and the control groups(P＜0.05). ConclusionThe humanepidermal stem cells can be selected by rapid attachment to collagen Ⅳ, and they can be expanded in culture if the appropriate conditions are maintained.