Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.