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A sample of reduced iron was separated by air elutriation into several fractions based on particle size; and the relative biological values (RBVs) of 3 fractions containing fine, medium, and coarse particles were determined with rats and chicks, using diets containing either skim milk powder or casein as the major source of protein. The RBV of reduced iron for rats was shown to be dependent on particle size by curative and prophylactic assay procedures, with the source of protein having no marked effect. In contrast to rats, chicks utilized reduced iron to a higher degree, especially the iron from coarse particles. Prophylactic assays with rats indicated that a 3-4 week feeding period from weaning at 21 days of age gave RBVs which were not significantly different from those obtained by repletion of hemoglobin of anemic animals. After 5 weeks on the test diets, however, the rats lost their ability to distinguish between coarse and medium particles of reduced iron when fed with a diet containing casein as the source of protein.