The effects of various structural modifications of human transferrin on its biologic activity have been studied by measuring three functions of the protein: (1) the iron-binding capacity of the molecule, (2) the binding of the protein to reticulocytes, and (3) the ability of the molecule to transfer iron to reticulocytes. By considering the reticulocyte-iron-transferrin interaction to be analogous to an enzyme-substrate reaction obeying Michaelis-Menten kinetics, the experimental data have been analyzed to yield apparent affinity constants of reticulocytes for these modified transferrins. Acetylation and carbamylation of up to 25% of the available amino groups results in a minimal decrease in iron-binding capacity. There is a marked stimulation of binding to noniron-transfer sites on the reticulocyte cell surface while the affinity for the physiologic iron-transfer sites is greatly impaired. The maximal rate of iron release to the cells is only slightly altered. Amidinated transferrin (84% modified), with an unaltered net charge, functions normally except for mild impairment of binding to iron-transfer sites. When transferrin is trinitrophenylated, binding to noniron-transfer sites occurs after 15% reaction while the iron-binding capacity does not decrease until 75% reaction. Extensively trinitrophenylated transferrin has an apparently normal binding affinity for iron-transfer sites on the cell surface and maximal iron transfer is greater than normal. However, the kinetic data, including competition experiments, suggest that there is a complex interaction of this modified transferrin with the reticulocytes. These studies demonstrate that the iron-binding capacity of transferrin is relatively insensitive to blockage of free amino groups and to the net charge of the protein. Binding of transferrin to reticulocyte iron-transfer sites is affected by both these parameters with net charge alterations being more important. Iron release to reticulocytes is normal or accelerated even with extensive protein modifications. The enzymatic removal of most of the carbohydrate molecules of the two heterosaccharide chains of transferrin causes no significant alteration in the biologic functions tested.