Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine-to-tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta2-microglobulin (beta2M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh-7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh-7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HFE was detected by western blot in HepG2 but not in Huh-7 cell membrane fractions. In WRL-68 cells expressing wild-type HFE, the HFE protein was largely found at the plasma membrane where it colocalizes with beta2M. On the contrary, the HFE-Y231del mutant, similarly to an exogenously expressed HFE-C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane-expressed beta2M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh-7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild-type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh-7 cells stably expressing exogenous wild-type HFE at the plasma membrane. Conclusion: Huh-7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HFE HC. (HEPATOLOGY 2010.)