To test the hypothesis that differences in duodenal iron absorption may explain the variable phenotypic expression among HFE C282Y homozygotes, we have compared relative gene expression of duodenal iron transporters among C282Y homozygotes [hereditary hemochromatosis (HH)] with and without iron overload. Duodenal biopsy samples were analyzed using real-time PCR for expression of DMT1, FPN1, DCYTB, and HEPH relative to GAPDH from 23 C282Y homozygotes, including 5 "nonexpressors" (serum ferritin < upper limit of normal and absence of phenotypic features of hemochromatosis) and 18 "expressors." Four subjects of wild type for HFE mutations without iron overload or liver disease served as controls. There was a significant difference in expression of DMT1 (P = 0.03) and DMT1(IRE) (P = 0.0013) but not FPN1, DCYTB, or HEPH between groups. Expression of DMT1(IRE) was increased among HH subjects after phlebotomy compared with untreated (P = 0.006) and nonexpressor groups (P = 0.026). A positive relationship was observed among all HH subjects regardless of phenotype or treatment status between relative expression of FPN1 and DMT1 (r = 0.5854, P = 0.0021), FPN1, and DCYTB (r = 0.5554, P = 0.0040), FPN1 and HEPH (r = 0.5100, P = 0.0092), and DCYTB and HEPH (r = 0.5400, P = 0.0053). In summary, phlebotomy is associated with upregulation of DMT1(IRE) expression in HH subjects. HFE C282Y homozygotes without phenotypic expression do not have significantly decreased duodenal gene expression of iron transport genes compared with HH subjects with iron overload. There is coordinated regulation between duodenal expression of FPN1 and DMT1, FPN1 and DCYTB, and FPN1 and HEPH and also DCYTB and HEPH in HH subjects regardless of phenotype.