We examined the relationship between cell proliferation and differentiation of cultured rat fetal and newborn hepatocytes isolated from various developmental stages. The albumin production rate increased along with cell growth under in vitro culture and became maximal two days after the growth cessation. AFP was secreted by both fetal and newborn hepatocytes with growth ability. Furthermore, the responses to HGF addition in fetal hepatocyte cultures were observed in terms of growth stimulation and down-regulation of the Met receptor. We also studied the changes in RB and liver enriched transcription factors (C/EBPs) for investigating the mechanism underlying proliferation and differentiation of fetal hepatocytes. Western blot analysis of hepatocytes taken from various gestation stages of rat liver showed that the expression of RB and C/EBPI} increased as gestation stage proceeded. When RB antisense S-oligonucleotide was added to the culture medium, proliferation and AFP expression increased, while C/EBPa and albumin expressions decreased. These results indicated that the tumor suppressor gene product RB had a profound role not only in cell proliferation but also hepatocyte differentiation.