To define the significance of the uptake step by unidirectional uptake of iron was measured on proximal small intestintestinal segments from Nembutal-anesthetized rats. The method employed a nonabsorbable marker of adherent incubating medium, and the studies were performed during the linear time period of instantaneous uptake. Uptake was linear over the concentration range 0.1-10 mM, from three iron complexes. The rate varied with the bioavailability of the iron in each complex. The Q10 was 1.0-1.3. Uptake rates were similar in all segments of small intestine. Uptake was not increased in segments from iron-deficient animals or rats with hemolytic anemia. Cobalt did not inhibit the uptake rate. These studies demonstrate that the uptake of nonheme iron by the small intestinal mucosal cell is by passive diffusion and further indicate that the uptake step does not contribute significantly to the mucosal control of iron absorption.