ABSTRACT Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the transferrin receptor 2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, while the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations which cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.