Iron absorption involves two carriers, one involved in the uptake of iron across the microvillus membrane of the enterocyte and the other in its transfer to the plasma at the basolateral surface. The uptake phase is thought to involve divalent metal transporter-1 (DMT1) which may move from the cytoplasm to the microvillus membrane under conditions of iron deficiency. To examine this possibility we used fasted animals previously fed an iron-deficient diet and then gavaged with iron. We measured the processes of iron absorption using in vivo gut sacs and correlated the changes observed with the intensity of DMT1 staining and gene expression in the duodenum. Fasting resulted in increased iron absorption, whereas gavage with iron decreased absorption. These changes were due to alterations in the uptake phase of absorption but not the transfer phase. There was also a highly significant correlation between the reduction in iron absorption, microvillus DMT1 staining and messenger ribonucleic acid (mRNA) expression. The loss of DMT1 from the microvillus membrane was not associated with an increase in cytoplasmic staining, suggesting that its loss was due to destruction of the carrier protein. It is concluded that DMT1 functional activity is determined by de novo synthesis and that the latter is regulated post-transcriptionally by enterocyte iron levels.