BACKGROUND/AIMS: We examined whether we could produce hepatocytes with further differentiated functions by promoting G1-S transition of the cell cycle in a butyrate-treated human hepatocyte cell line that we have established. Since cyclin E is the main regulatory protein controlling the G1-S transition, in this study we have overexpressed cyclin E to promote the G1-S transition in human hepatocytes. METHODS: A conditional cyclin E-overexpressing cell line was established by transfecting human cyclin E cDNA into a human hepatocyte. The percentage of cells in the S, G0-G1 and G2/M phases of the cell cycle was determined using flow cytometry. Albumin-secreting and ammonia-detoxifying capacity, and the transcription and activity of ornithine transcarbamylase of the cells were analyzed to evaluate hepatocyte-specific functions. RESULTS: The G1-S transition of the cell cycle was significantly accelerated in the cyclin E-overexpressing cells as compared to the control cells. Upon butyrate treatment, the cyclin E-overexpressing cells exhibited a significantly increased albumin-secreting and ammonia-detoxifying capacity as compared to the control cells. In particular, the ornithine transcarbamylase activity was increased in these cells. CONCLUSIONS: These results suggest that the promoted G1-S transition augments the hepatocyte-specific functions during the butyrate-induced differentiation process of human hepatocytes.