We have previously shown that coexpression of c-myc and TGF-α in the liver results in accelerated replicative senescence and promotes tumor development in young adult transgenic mice. Here we describe the characteristics of hepatocyte proliferation in primary cultures established from 10-week-old control, c-myc and c-myc/TGF-α transgenic mice. A variety of cellular and functional changes occurred in the transgenic livers at this age including enhanced polypoidization and impairment of hepatic functions. Control mouse hepatocytes demonstrated a high level of DNA synthesis in serum-free medium with a maximum at day three in culture at which time 70% of the cells were in S phase. In contrast, DNA synthesis peaked one day later and was reduced by 50% in the cultured c-myc and c-myc/TGF-α hepatocytes. Also, higher frequency of apoptosis was observed in the transgenic hepatocytes. However, in hepatocytes isolated from c-myc/TGF-α mice, which show early appearance of preneoplastic lesionsin vivo,the DNA synthesis continued for 6 days in culture in contrast to a sharp decrease in the labeling index of control and c-myc hepatocytes after 3–4 days in culture. The results suggest that proliferative features of the transgenic hepatocytesin vitroreflect the general properties of these cellsin vivoand thus may provide a model for studies on senescence and transformation of hepatocytes.