Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interaction is not understood. We tested the hypothesis that divalent metal-ion transporter-1 (DMT1) the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate Ca-45(2+) uptake. Instead, we found that Ca2+ blocked the Fe2+-evoked currents and inhibited Fe-55(2+) uptake in a noncompetitive manner (K-i approximate to 20 mM). The mechanism of inhibition was independent of voltage and did not involve intracellular Ca2+ signaling. The alkaline-earth metal ions Ba2+, Sr2+, and Mg2+ also inhibited DMT1-mediated iron-transport activity. We conclude that Ca2(+) is a low-affinity noncompetitive inhibitor but not a transported substrate of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability. (C) 2010 Elsevier Inc. All rights reserved.