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Background: Iron is crucial for growth and development, but excess iron is harmful. Neonatal mice have elevated concentrations of circulating iron, but the source of this iron is unclear. This lack of understanding makes it difficult to optimize early life iron balance., Objectives: Identify the origins of neonatal tissue-specific iron pools using dietary manipulation and cross-fostering murine models., Methods: To determine whether tissue-specific neonatal iron was primarily acquired during gestation or after birth, pups born to iron-sufficient or iron-deficient dams were cross-fostered, and tissues were harvested at postnatal days 3-5 to measure iron content. A separate set of female mice were fed a diet enriched with the stable iron isotope 57 ( 57 Fe) for 4 generations to replace naturally abundant liver iron isotope 56 ( 56 Fe) stores with 57 Fe. To quantify the proportions of neonatal iron acquired during gestation, pups born to dams with 56 Fe or 57 Fe stores were cross-fostered, and tissues were harvested at postnatal day 3-5 to determine 56 Fe: 57 Fe ratios by inductively coupled plasma mass spectrometry. Finally, to quantify the proportion of neonatal iron acquired from the maternal diet, female mice with 56 Fe or 57 Fe stores switched diets upon mating, and pup tissues were harvested on P0 to determine 56 Fe: 57 Fe ratios by inductively coupled plasma mass spectrometry., Results: Perinatal iron deficiency resulted in smaller pups, and gestational iron deficiency resulted in lower neonatal serum and liver iron. Cross-fostering between dams with 56 Fe and 57 Fe stores demonstrated that ≤70% of neonatal serum, liver, and brain iron were acquired during gestation. Dietary manipulation experiments using dams with 56 Fe and 57 Fe stores showed that over half of neonatal serum, liver, and brain iron were from the dam's gestational diet rather than preconception iron stores., Conclusions: This study provides quantitative values for the sources of neonatal iron, which may inform approaches to optimize neonatal iron status., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)