Primary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml epidermal growth factor in a 5% CO2:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.