A cell line, HuH-33, was established in vitro from a patient with hepatocellular carcinoma (HCC), who had been treated with some chemotherapeutic agents. The chromosome number was widely distributed in 6888. This cell line has grown slowly, exhibiting a doubling time of approximately 150h, was intransplantable into nude mice, and secreted alpha-fetoprotein, albumin, β2-microglobulin, ferritin, tissue peptide antigen and extracellular matrix materials. HuH-33 was polygonal or spindle shaped, but it was keratin-positive and desmosome-like structures were observed with transmission electron microscopy, suggesting an epithelial origin of HuH-33.