We describe the establishment and characterization of a novel hepatoma cell line. This cell line, designated RBHF-1, was established from a hepatocellular carcinoma of a 67-yr-old man with a history of genetic hemochromatosis. At this writing, the cells have been maintained in RPMI-1640 tissue-culture medium and fetal calf serum without any additional supplements for 30 mo. The cells form colonies on soft agar and are not tumorigenic in nude mice. The cell line is polymorphic and displays characteristics of mature hepatocytes by synthesizing albumin, alpha 2-macroglobulin, fibronectin and alpha-fetoprotein. Cytogenetic analysis shows multiple chromosomal aberrations, with a consistent deletion in the long arm and deletions or rearrangements in the short arm of chromosome 1. There is no evidence for hepatitis B or hepatitis C virus infection of the cell line. The cells contain no detectable intracellular iron after staining with Perls' stain. Unlike other hepatoma cell lines, there is no detectable binding of epidermal growth factor to RBHF-1 cells. This is the first cell line to be established from a patient with hemochromatosis, and it provides a potentially important model for the study of hepatocyte transformation in association with iron overload.