Objective：To identify the maintenance of an undifferentiated state of mouse alveolar epithelial typeⅡ（ATⅡ） cells by the co-culture of feeder cells（mouse embryonic fibroblast cells，MEFs）. Methods：The mouse lung single cells were prepared by the com－bination use of dispase，collagenase and DNase I. ATⅡ cells were purified by immune adherence. The culture medium was high glu－cose dulbecco modified eagle medium（HG/DMEM） addicted with 10% fetal bovine serum（FBS）and antibiotics. ATⅡ cells were co-cultured with MEFs and were identified by immunofluorescence staining of pulmonary surfactant protein C（SPC） and Aquaporins 5（AQP5）. Results：（1.7-2.0）×107 cells per mouse lung was obtained. ATⅡ cells can maintain their undifferentiated state until the fourth generation when being cultured on feeder cells，but will differentiate into alveolar AT Ⅰ cells when being cultured alone. Conclusion：MEF cells can maintain the undifferentiated state of ATⅡ.