Abstract: The aim of the present study was to establish the methods for isolation and identification of mouse hepatocytes with high purity and viability,and to evaluate their purity and biological activity.The mouse hepatocytes were isolated and purified by using two-step in-situ-circulating perfusion method in combination with multi-round of low speed differential centrifugation.Hepatocytes were primarily cultured in adherent culture medium and their viability were evaluated by trypan blue exclusion.PAS reaction and immunofluorescence assay were used to examine their glycogen synthesis ability and CK18 expression level,respectively.The results showed that a total of 1.5×10^6 hepatocytes with >97% of motility rate could be harvested from individual mice in this study.The primary adherence of hepatocytes was observed after 6 h of inoculation under inverted microscope.The adherent cells grew well 72 h post inoculation.The size of the hepatocytes was enlarged with irregular shape,and they were connected with the surrounding cells in island or strip-like junctions.Glycogen deposits in form of purple-red particles were found in hepatocytes in which CK18 was uniformly distributed.PAS reaction in combination with immunofluorescence staining for CK18 demonstrated the high-purity (>90%) of hepatocytes.In conclusion,the mouse hepatocytes isolated by using the established method in this study had high harvest yield and viability,and the cells cultured in adherent culture medium had high purity,providing a foundation for further investigation on metabolism and cytotoxicity of hepatocytes.