Divalent metal transporter I (DMT1) is thought to be involved in transport of iron across the apical cell membrane of villus duodenal cells. To determine its role in hereditary hemochromatosis (HH), we used ₂-microglobulin knockout (B2M/) mice that accumulate iron as in HH. The B2M/ and control C57BL/6 (B2M+/+) mice were fed diets with different iron contents. Increasing the iron availability increased plasma iron levels in both B2M+/+ and B2M/ mice. Reducing the iron availability decreased the plasma iron concentration in B2M+/+ mice but was without effect on plasma iron in B2M/ mice. DMT1 was not detectable in mice fed normal or iron-loaded diets when using immunohistochemistry. In Western blots, however, the protein was consistently observed regardless of the dietary regimen. DMT1 expression was increased to the same extent in B2M+/+ and B2M/ mice when fed an iron-poor diet. In both strains of mice fed an iron-poor diet, DMT1 was evenly distributed in the differentiated enterocytes from the base to the tip of the villi but was absent from the crypts of Lieberkühn. These data suggest that the observed effects were due to the state of iron deficiency in mucosal cells rather than genetic defect.