Iron transport across polarized intestinal epithelium was studied by using Caco-2 cells grown in bicameral chambers. When cells were grown under conditions of low, normal, or high iron concentration not only was the iron content of the cells markedly altered but the low iron cells exhibited a nearly 2-fold increase in transepithelial electrical resistance (TEER). 59Fe uptake from the apical surface into cells and transport into the basal chamber was affected both by the valency of the iron and the iron status of the cells. Uptake from 59Fe(II)-ascorbate was about 600 pmol 59Fe/h per mg protein, increased about 2-fold in low iron cells, and was about 13-200-fold greater than uptakes from 59Fe(III) chelated to nitrilotriacetic acid, BSA, or citrate. Transport into the basal chamber from 59Fe(II)-ascorbate was 3.7 +/- 1.7 pmol/h per cm2 for Fe-deficient cells vs. 0.72 +/- 0.1 pmol/h per cm2 for normal-Fe cells and from 59Fe(III)-BSA 1.1 +/- 0.2 pmol/h per cm2 vs. 0.3 +/- 0.03 pmol/h per cm2 for deficient vs. normal iron cells, respectively. The greater transport of iron both from Fe(II) and in iron deficient cells supports the use of the Caco-2 cells as a model for iron transport.