Non-transferrin bound iron (NTBI) in serum is cleared rapidly by hepatocytes, although the mechanisms of NTBI uptake by hepatocytes are poorly understood. Dietary iron is transported into intestinal enterocytes by divalent metal transporter 1 (DMT1), which also transports iron from transferrin receptor 1 (TfR1)-mediated recycling endosome to intracytoplasm. We made an antiserum against human DMT1 protein derived from mRNA with the iron responsive element (IRE). The DMT1 detected by the antiserum was mainly observed in the membranes of duodenal enterocytes and enterocyte carcinoma (Caco2) cells, whereas DMT1 in normal liver and hepatoma (HLF) cells, was preferentially located in cytoplasm but weakly on cell surface. In addition, iron-depleted HLF increased membrane expression of DMT1, suggesting that the intracellular iron concentration regulated the DMT1 expression in hepatocytes via the iron regulatory protein (IRP)/IRE system. DMT1 overexpressing HLF by DMT1 cDNA transfection expressed DMT1 in both cytoplasm and cell membrane. Although these cells did not change TfR-dependent iron uptake, they took up a significant amount of ferrous iron. These results indicate that the DMT1 plays an important role in transporting NTBI into cells.