Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2; Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin. Conclusion: These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8. (HEPATOLOGY 2009.)