Human hepatocytes were isolated with EGTA and collagenase perfusion method from eleven different liver tissues in this experiment. The viability of freshly isolated human hepatocytes was between 65% and 85%, and the primary hepatocytes could survive in the collagen G coated culture plate for 2 to 4 weeks. During this time, the hepatocytes presented the typical polygonal shape, and expressed epithelial cell markers, CK 8 and CK 18. They also expressed albumin, ASGPR, alpha1-antitrypsin and metabolized lidocaine, which are the characters of differentiated hepatocytes. Human intrahepatic BECs were isolated with immunomagnetic separation method from ten different liver tissues. The BECs could proliferate in the culture medium (containing EGF and HGF) for at least 4 weeks, and some BECs had proliferated for more than 18 weeks and been passaged for 12 times. Primary BECs were small and oval, and they expressed biliary cell markers (CK 7 and CK 19) and epithelial cell markers (CK 8 and CK 18), while they did not express desmin (marker of hepatic stellate cell), factor VIII (marker of endothelial cell). The further characterization repeatedly found that proliferating BECs not only kept on expressing CK 7, CK 19, CK 8 and CK 18, but some also expressed c-kit, chromogranin-A, albumin, AFP, vimentin (after passage), alpha1-antitrypsin, which are the markers of liver stem cells. Some BECs had the morphology of hepatocytes and could survive for more than 4 weeks without the change of culture medium. Double-fluorescent immunostaining identified the biliary origin of these cells. All of these phenomena suggested that there were some liver stem-like cells co-purified with intrahepatic BECs in vitro. Liver stem cells might be residents among intrahepatic BECs and activated in vitro by the growth factors. This finding supported the hypothesis that the human biliary tree may harbour liver stem cells, and the liver stem cells could be co-purified with intrahepatic BECs.