A simple method to culture rat liver parenchymal cells is described. Minced liver tissues were dissociated with no pre-perfusion by a newly developed enzyme, bacterial neutral proteinase, and cultured in the medium consisting of 10% fetal calf serum and 90% of a new synthetic medium, DM-153. By the adequate technique of primary dissociation and first subcultivation, parenchymal cells were selected. Cultivation in arginine-free medium was also useful in selecting them. The cultured cells exhibited activities of certain enzymes similar to those of liver cells in vivo. Cell strains have been successively established from liver tissues of embryo, suckling and adult rats. Liver cells, however, cultured in this way, can also be used for experiments in the early stage of serial cultivation.