The distribution and quantitation of the iron-binding proteins of rat small intestinal mucosa was studied, in iron-deficient and replete animals, to explore their role in the absorption of iron. Adsorption (mucosal uptake) of iron in in situ ligated loops of small intestinal mucosa was found to be uniform throughout the length of the small intestine whereas absorption (carcass uptake) showed a steep decreasing gradient from the duodenum to the ileum. The disrupted, in vivo labeled mucosal cells were fractionated by isopycnic centrifugation and transferrin and ferritin were quantitated by radioimmunoassay. Transferrin derived from mucosal cells was shown to have a higher affinity for the antibody than transferrin in serum. Of the transferrin present in the mucosal extract, only a portion could be accounted for by contamination from the serum; the proteolysis resistant and intrinsic transferrin may be mucosal cell specific. Transferrin was found in similar amounts in all regions of the small intestine, was not affected by iron loading but doubled in response to iron deficiency. Mucosal ferritin was found in greater amounts in the iron-absorbing areas of the intestine, increased in the duodenum of iron-loaded animals, and decreased in iron-deficient animals. The incorporation of newly absorbed radioiron into ferritin was only found in iron absorbing regions and was completely inhibited by colchicine and cytochalasin-B, suggesting that ferritin was loaded with iron at the point of iron absorption and that the process is associated with vesicle movement and not simple diffusion. Transferrin and ferritin-specific immunoabsorption and also gel filtration established that no other soluble iron binding proteins were involved in absorption.