Transfection methods for primary cultured mouse hepatocytes were examined. Of four conventional transfection methods examined, involving use of DEAE-dextran, calcium phosphate, cation-liposomes (lipofection), and cation-multilamellar liposomes, only cation-liposomes induced highly efficient transfection into primary cultured mouse hepatocytes. The highest transfection rate reached more than 60% of the total cells. Three other commonly used cell types (CHO-K1, COS-1, 3T3-L1) were also tested as target cells, but highly efficient transfection was observed specifically in primary cultured mouse hepatocytes. The transfection remained at a high level from 6 to 48h after the start of incubation with the cation-liposome-DNA complex in the absence of serum, and the transfection rate decreased in inverse relation to the increase in cell density. The transfection was inhibited by free low density lipoprotein (LDL), EDTA, and an endocytosis inhibitor, cytochalasin B. These data suggest that the transfection is mediated not only by membrane fusion, as is generally accepted, but also by endocytosis. This information should be useful for research in hepatocyte biology and the development of gene therapy. As one of the applications, simple and successful immunization was achieved by administration of hepatocytes transfected with murine adhesion molecule, integrin VLAβ1 subunit, genes into a Syrian hamster.