Divalent Metal Transporter 1 (DMT1) is a transmembrane transporter located at the apical membrane of enterocytes and implicated in the duodenal uptake of iron. Results from expression experiments in Xenopus oocytes indicate that DMT1 can mediate transport of a wide range of divalent metals other than iron. The aim of the present study was to examine the effect of iron treatment on the uptake and transepithelial movement of 63Ni and to correlate that to DMT1 messenger RNA (mRNA) levels in human intestinal Caco-2 cells. Twenty-one days after confluent Caco-2 cell monolayers were treated for 1 or 3 days with medium supplemented with Fe(NTA)2 or control medium and 63Ni transport and DMT1 gene expression were measured at both time points. Functional effects of the iron treatment were assessed by examining uptake and transepithelial movement of 59Fe. Iron treatment resulted in decreased DMT1 gene expression which correlated well with the uptake of 59Fe and 63Ni into fully differentiated Caco-2 cells. This indicates that DMT1 is responsible for the apical transport of these metals in the intestinal epithelium and suggests that adequate iron intake and status will limit nickel absorption.