The subcellular localization of intraduodenally administered 59Fe has been studied in control, pregnant, and nonpregnant rats absorbing iron at different rates. Binding of iron to the particulate fractions of mucosal homogenates produced by centrifugation at 500, 10,000, and 100,000 X g did not vary among groups despite significant alterations in iron transport rates. Study of the 10,000 X g supernatant fluid showed that increased iron transport in iron-deficient and day 20- to 21-pregnant rats was associated with an increase in 59Fe binding to a particulate fraction of the mucosal homogenate. Although only 10 to 15% of the total mucosal 59Fe was present in the 100,000 X g supernatant in all groups, significant differences were demonstrated, among groups in the binding of iron to the three proteins present in this supernatant fraction. The proteins have been identified as ferritin, transferrin, and a protein of lower molecular weight than transferrin. Increased iron absorption was associated with a reduction in ferritin 59Fe binding and an increase in 59Fe bound to protein 3. Conversely, reduced iron absorption, was associated with increased ferritin 59Fe content and decreased protein 3 59Fe content. By studying day-21 pregnant rats 5 and 18 hr posthysterectomy it was possible to demonstrate a 12- to 18-hr delay in the intestinal mucosal protein response to alterations in the stimulus to iron absorption.