A human hepatocyte line (HHY41) was established from normal human liver tissue. This cell line was derived from a primary culture of human hepatocytes maintained between two layers of collagen gel for 4 weeks. It differs from other human hepatocyte lines in that transfection with the simian virus 40 gene was not used for cellular transformation and nonhepatocellular coculture cells were not present. HHY41 cells have proliferated freely in serum and hormone-supplemented medium after more than 1 year in continuous culture, exhibiting typical morphological characteristics of hepatocytes. HHY41 cells retain glucose-6-phosphatase activity. They also retain the ability to secrete liver-specific proteins such as albumin, transferrin, and alpha-fetoprotein. Northern blot analysis confirmed the presence of albumin mRNA. Cytochromes P450 induced by polycyclic aromatic hydrocarbons are maintained in these cells. Detection of cell surface antigens revealed that HHY41 cells express alpha 1 beta 1-integrin, which is expressed by normal hepatocytes and not by bile duct epithelial cells. High-molecular-weight cytokeratin, a marker for bile duct cells, is also absent in HHY41. Cytogenetic analysis showed hyperdiploid karyotype with a consistent deletion in the short arm of chromosome 1. HHY41 can be considered a new human hepatocyte line which retains liver-specific functions of differentiated hepatocytes. Derived from normal liver tissue, not a hepatocellular carcinoma, it provides a new model system for studying the regulation of cell growth and differentiated functions in human hepatocytes.