Technical Abstract: Sodium Iron EDTA (NaFeEDTA) has superior iron bioavailability especially in foods containing iron absorption inhibitors. However, mechanisms involved in the absorption and subsequent partitioning of iron complexed with EDTA are poorly understood. Our objectives were to compare retention and tissue distribution of iron administered to rats either as FeSO4 or NaFeEDTA, either orally (OR) or subcutaneously (SC). Weanling rats were fed purified diets supplemented with either FeSO4 or NaFeEDTA for 7 days. They were then given a meal containing 59Fe labeled FeSO4 or NaFeEDTA, or they were injected SC with these forms of radiolabeled Fe. 59Fe retention was measured by whole body counting. Urine was collected and counted at 24-hour intervals throughout the counting period. Tissue samples were analyzed for nonheme iron and 59Fe activity. Absorption of iron from FeSO4 or NaFeEDTA was similar (61.7% and 57.2% respectively). Seventy-seven percent of the injected Na59FeEDTA was excreted in the urine within 48 hours, whereas only 0.5%, 0.8%, and 1.4%, of the injected 59FeSO4, oral 59FeSO4 and oral Na59FeEDTA, respectively, was excreted in the urine. Nonheme iron content was lower in the liver and spleen, by 56.8% and 28.4%, respectively, among rats consuming the NaFeEDTA diet compared to rats fed FeSO4. We conclude that iron is dissociated from EDTA prior to or during intestinal absorption and that some fraction of the dissociated EDTA is absorbed separately from the iron.