A combination of biochemical quantitation and immunohistochemistry has been used to examine in detail transferrin receptor distribution and expression in the rat small intestine and its relationship to iron absorption. Receptor numbers were quantitated by transferrin binding to preparations of basolateral or brush-border membranes. Receptors were demonstrated on the basolateral membranes of the gut cells, but not on the brush-border fraction. Apotransferrin demonstrated little binding to basolateral membranes at physiological pH. Dietary or parenteral iron loading of animals produced a significant decline in transferrin binding, whereas binding was increased in iron deficiency. These data were confirmed by immunohistochemical studies using a monoclonal antibody to the transferrin receptor. When iron absorption was increased threefold following acute hemolysis and without a decrease in body iron stores, there was no change in transferrin receptor number. These data indicate that intestinal transferrin receptors may be regulated by body iron stores but suggest that they are not directly involved in iron absorption.