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Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study wa. to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF101, which contains no peptides other than recombinant human transferrin and insulin. As a result, air immortal human hepatocyte cell line (OUMS-29) having li er-specific functions was established from one of the 13 clones. Expression of CYP 1A1 arid 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) arid AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependent by these polycyclic aromatic hydrocarbons. This cell line is expected to he instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis. drug metabolisms of liver cells, and hepatic toxicology,.