Since human hepatocytes are available only in Limited number, the development of a serum-free culture system for long-term cultivation of differentiated and functional hepatocytes is of great importance. Here we describe the culture of human hepatocytes in a chemically defined serum-free medium for up to 5 weeks. Cell morphology was assayed by light and electron microscopy and revealed a well-preserved cellular morphology. Marker proteins for epithelial and bile duct cells, cytokeratin (CK) 18 and 19, and liver-specific proteins, Like phosphoenolpyruvate carboxykinase-2 (PCK2) and serum proteins, were expressed. fiver-enriched transcription factors CCAAT/enhancer binding protein alpha (C/EBP alpha) and hepatocyte nuclear factor-4 (HNF-4), cytokine and mitogen activated factors (nuclear factor kappa B) NF kappa B, and, activator protein-1 (AP-1) were maintained and active for several weeks in our cultures. In summary, our serum-free culture system allows the culture of differentiated human hepatocytes for several weeks. It may serve as a model system for metabolic, pharmacologic-toxicologic studies, and studies on human pathogens under defined chemical conditions.