Eight permanent human hepatocellular carcinoma (HHC) cell lines were established from 8 individual patients by the use of aspirated needle biopsy specimens (smaller than 0.1 ml in size). The cells grew in clustered form and retained intercellular junctions and canaliculi resembling bile canaliculi. The presence of secreted human alpha-fetoprotein and human albumin was detected in the cultured medium. Hepatitis B surface (HBs) antigen was not found on these cells. Implantation of the cells into athymic mice was followed by the growth of hepatocellular carcinomas and the appearance of human alpha-fetoprotein in the mouse serum. Chromosome analysis of three of the cell lines showed hyperdiploidy in two of them and hypotetraploidy in the other. Enzyme analyses of culture medium and cell homogenates have detected some enzymes characteristic of liver tissue such as gamma-glutamyl transferase, sorbital dehydrogenase, alkaline phosphatase, glutamate dehydrogenase, as well as aspartate and alanine transaminase. These tumor cells have been continuously maintained in culture for over 6 years with no significant changes observed.