In the present study, we showed the role of the liver-enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We used primary rat hepatocytes cultured in modified L-15 medium. The cells proliferated and, after the addition of 2% dimethyl sulfoxide (DMSO) from day 4, they stopped growing and gradually differentiated. During hepatic proliferation, expression of hepatocyte nuclear factors (HNF)1alpha, HNF4, C/EBP alpha, and C/EBP beta mRNAs was depressed, whereas that of HNF3alpha and HNF3beta transcripts was enhanced. After the addition of DMSO, the expression of HNF1alpha, HNF3gamma, and HNF4 returned to the level in isolated cells and HNF1beta mRNA expression gradually increased. However, expression of C/EBP alpha and C/EBP beta mRNAs was partially recovered. The mitoinhibitory agents, IL-1beta, IL-6, TGF-beta, and activin A, were examined to determine whether they could induce differentiation of proliferating hepatocytes as shown in cells treated with DMSO. Although these factors inhibited cell growth, the cells did not differentiate. The expression pattern of HNF3gamma mRNA was quite different in the cells cultured with DMSO and those cultured with cytokines. Therefore, hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription factors. Thus, expression of HNF3gamma, C/EBP alpha, and C/EBP beta may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1alpha, HNF1beta, HNF3alpha, HNF3beta, and HNF4 as well as suppression of C/EBP delta.