Technical Abstract: Iron uptake from radiolabeled ferrous sulfate, ferrous ascorbate, ferrous bis-glycinate, ferric chloride, ferric citrate and ferric EDTA by Caco-2 cells with different iron status, and in the presence of divalent metal cations, was compared. Supplemental iron, added to the cell culture medium at 20 umol/L 72 h prior to the uptake experiments, elevated the iron status of Caco-2 cells by increasing ferritin synthesis 100-fold, increasing HFE gene expression by 40%, and decreasing DMT-1 gene expression by 28%. In cells receiving supplemental iron, there were significant decreases in uptake from radiolabeled ferrous ascorbate and ferrous bis-glycinate, but not from ferric compounds, when compared to cells receiving no supplemental iron. Regardless of the iron status, ferrous ascorbate showed the highest percent uptake, followed by ferrous bis-glycinate, whereas uptake from all other forms of iron was significantly lower. Iron uptake from both salts and chelates was markedly inhibited in the presence of 100-fold molar excess of Co(II) and Mn(II) cations. The inhibition was more pronounced in ferrous iron uptake than ferric iron uptake, with ferrous ascorbate showing the greatest percent reduction (-90%). Our results suggest that ferrous iron is the predominant form of iron taken up by intestinal epithelial cells, and the DMT-1 pathway is the major pathway for iron uptake. Iron uptake from ferrous bis-glycinate and ferric EDTA appears to follow the same pathway as uptake from iron salts.