Objective assessment of the differentiation grade of hepatocellular carcinomas (HCCs) is important for evaluation of the pathological diagnosis, prognosis and therapeutic treatment. Differentiation of hepatocytes is reflected by their expression of hepatic functional proteins in the mouse embryo, and liver-enriched transcription factors (LETFs) have been shown to regulate hepatic functional genes strictly. Previous reports demonstrated that the level of LETF expression is altered in HCC or preneoplastic nodules compared with noncancerous tissues. Therefore, LETF expression levels might be useful as a measure of HCC maturation. In this study, to clarify the correlation between the expression of LETFs and the differentiation grade of HCCs, we performed a quantitative analysis of the mRNA expressions of HNFs and C/EBPα using real-time reverse-transcription PCR and immunocytochemical analysis for hepatic functional proteins in twelve cell lines. Furthermore, we examined orthotopic transplantations of the HCC cell lines in C.B-17/Icrj-scid/scid mice and characterized the histologic and cytologic differentiation of the tumors that developed. Our results showed that comprehensive expressions of HNF-3β, HNF-4α, HNF-1α, and C/EBPα were specific to HCCs with well-differentiated function and morphology. Furthermore, among these four transcription factors, HNF-4α and HNF-1α expressions showed synchronism and had a close relation with HCC differentiation. These in vitro results were confirmed in tumors developed in SCID mice in vivo. These findings suggested that HNF-4α and HNF-1α are useful markers to assess the degree of HCC differentiation, which we suggest could be evaluated objectively by the quantitative analysis of HNFs and C/EBPα in HCCs.