Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.