We have previously reported a medium that supports the continuous growth of hepatocytes without their losing replicative potential and differentiation capacity for an extended period. The medium contains four key substances in addition to fetal bovine serum, that is, epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate, and dimethyl sulfoxide. When a nonparenchymal cell fraction containing small hepatocytes and nonparenchymal cells was cultured in this medium, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. The growth potential of small hepatocytes was variable among the cells, the highest case being that of a single cell that produced a colony containing over 100 cells in 10 days. When a hepatocyte was allowed to divide for 105 days, it produced a colony of approximately 0.2 mm2, which contained approximately 1,700 hepatocytes, indicating that the cell divided more than 10 times. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver in vitro.