The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.Iron is a trace element that is essential for life, since it plays a critical role in many biochemical and physiological mechanisms. As a consequence, nature has developed an array of elaborate processes for the absorption, storage, and transport of iron within the body, and a homeostatic balance between these mechanisms is essential for good health.The vast majority of dietary non-heme iron is absorbed in the duodenum, where it is most soluble due to the acidic environment. Bioavailable iron is always in the ferrous (Fe 2ϩ ) state, but most ingested iron is in the ferric (Fe 3ϩ ) form. Reduction of Fe 3ϩ to Fe 2ϩ can be promoted by the action of a putative ferric reductase (1) and reducing components in the diet, such as ascorbate (2). The regulation of iron absorption from the diet by the small intestine is of crucial importance in determining body iron status, and consequently, a great deal of research interest has focused on the cellular mechanisms involved in iron accumulation. This has culminated in the expression cloning of an iron transporter from rat duodenum (3) and the positional cloning of the human homologue, Nramp2 (4). In the Caco-2 cell model of human small intestinal enterocytes Nramp2 mRNA is down-regulated by increasing cellular iron status (5), suggesting a role in iron homeostasis. In addition, in both microcytic anemic (mk) mice (4) and Belgrade rats (6), there is a defect in intestinal iron transport that has been mapped to the gene encoding the protein Nramp2. Taken together these data suggest to us that Nramp2 should be expressed on human enterocyte plasma membranes and function as an iron transporter. Our present study has tested this hypothesis, and accordingly, we present evidence that human enterocytes absorb iron across their apical membrane in a pH-dependent fashion. Using cells loaded with the fluorescent dye BCECF, 1 we show that there is a concurrent intracellular acidification induced by iron in the bathing medium. With an antibody generated against Nramp2, we demonstrate the expression of a 66-kDa apical membraneresident p...