Objective:To explore the method of isolation,cultivation and identification of adult rats pancreatic duct stem cells in vitro. Methods:The pancreas of adult rats were digested by injecting collagenase V solution and purified by removing the islets with Ficoll density gradient centrifugation. The islet-depleted tissue was cultured in RPMI1640 with 10% fetal calf serum,and the medium was supplemented with EGF and bFGF for 7 days,then the cells could form a monolayer. 0.25% Trypsin-EDTA were used to detach the cells, which were serial-subcultivation. Cells from the second passage(P2) were used to detect the expression of CK19,Pdx-1,Nestin,insulin and glucagon with methods of immunofluorescence staining and RT-PCR. Results:Through collagenase solution injecting and digesting the pancreatic duct,and depleting the islet tissue after Ficoll density gradient centrifugation,the pancreatic duct cells could get a better purification. The results of immunofluorescence staining revealed that the expression of CK19,Pdx-1 and Nestin of P2 cells were positive, and the rate of positive cells were(88.6 ± 6.2)%,(84.6 ± 8.6)% and (79.3 ± 10.5)%,respectively,while insulin and glucagon staining were negative. The results of RT-PCR demomstrated that the cells also expressed CK19,Pdx-1 and Nestin. Conclusion:This method can isolate the pancreatic duct cells well,and the cells obtained have the character of stem cells through identification.