The recent discovery of HFE, the MHC-Class-I-like gene mutated in up to 90% of patients with hereditary haemochromatosis, and the gene encoding the Nramp2/divalent metal transporter-1 (DMT-1) implicated in ferrous iron transport holds promise for a greater understanding of human iron metabolism. Since the HFE protein can be crystallized as a ternary complex with the transferrin receptor and iron-saturated transferrin, and DMT-1 expression is up-regulated in hereditary haemochromatosis, these proteins are likely to interact in a common pathway for human iron homeostasis. To investigate the cellular interactions between the cognate proteins encoded by these genes, we generated a panel of rabbit and avian antisera from human HFE and DMT-1 derived peptides. The antibodies were characterized by ELISA reactions and Western immunoblotting. Immunohistochemical staining showed that DMT-1 protein localized to the brush border of human duodenum where it is predicted to serve as the principal transporter of ferrous iron from the intestinal lumen. In the human cell lines, Caco-2 (small intestinal phenotype upon differentiation) and K562 (erythroleukaemic) HFE, in the presence of iron-saturated transferrin, co-localized with transferrin receptors in an early endosome compartment using confocal immunofluorescence microscopy. This interaction may be critical in small-intestinal crypt cells which express HFE, where it may function to modulate their intrinsic iron status thereby programming iron absorption by DMT-1 in the mature enterocyte. In undifferentiated Caco-2 cells, DMT-1 localized to a discrete late endosome compartment distinct from that occupied by HFE where, in addition to brush-border iron uptake, it may function to regulate the availability of iron delivery to intracellular iron pools. Disruption of the HFE gene as a result of mutations associated with hereditary haemochromatosis may thus impair homeostatic mechanisms controlling iron absorption within the small-intestine epithelium by a direct interaction with transferrin receptors and by subsequent alteration of DMT-1 expression. Identification of the molecular interactions of HFE with DMT-1 and other key components of the iron transport pathway has implications for a mechanistic understanding of the pathophysiology of human iron storage diseases as well as the regulation of normal iron balance.