The effect that metal attachment to apotransferrin exerts on the binding affinity of reticulocyte surface receptors sites for transferrin has been studied using two methods: (1) by direct binding studies using iodinated transferrin and (2) by competition experiments in which the ability of the test transferrin to impair reticulocyte Fe3+ uptake from 59Fe3+-transferrin was determined. While the absolute values for the dissociation constants for transferrin binding to reticulocytes varied somewhat between preparations of reticulocytes, the relative affinities of the various transferrins were constant. Typical apparent dissociation constants obtained by direct binding studies were 0.5 mg/ml for apo- and Cu2+-transferrin, 0.28 mg/ml for Cr3+-transferrin and 0.13 mg/ml for di-Fe3+-transferrin. Di-Fe3+-transferrin had a dissociation constant that was 30% lower than mono-Fe3+-transferrin. Typical inhibitor constants obtained by competition experiments were 1.06 mg/ml for apotransferrin, 0.65 mg/ml for Cr3+-transferrin, 0.19 mg/ml for mono-Fe3+-transferrin and 0.14 mg/ml for di-Fe3+-transferrin. These data show that the binding of transferrin to its cellular receptor site is greatly influenced by the attachment of metals to the protein. This effect is primarily related to the particular metal that is bound and is less dependent on whether 1 or 2 atoms of metal are bound per mole of protein. Data obtained from gel filtration experiments indicates that metal attachment to apotransferrin results in different conformational states of the protein. On a column of Sephadex G-150, Fe3+-transferrin was slightly more retarded than either apo- or Cr3+-transferrin, suggesting that the Stokes radius of Fe3+-transferrin is smaller than that of the other molecules. Whether metal binding to apotransferrin affects the binding of the protein to reticulocyte receptor sites by inducing a conformational change in the protein or by directly participating in the binding cannot be determined from these data.