At pH 5.4 apotransferrin (iron-free transferrin) binds to cell-surface transferrin receptors to the same extent and with the same affinity as does diferric transferrin at pH 7.0. Apotransferrin is quickly dissociated from its receptor when the pH is raised to 7.0. These and other results strongly support a simple model that explains the cycling of transferrin during a single cycle of receptor-mediated endocytosis. Diferric transferrin binds to cell-surface receptors, and the transferrin-receptor complex is endocytosed. The pH of the endocytic vesicle is lowered to 5.5 or below; this causes dissociation of iron from the transferrin-receptor complex, but apotransferrin remains bound to its receptor. The iron remains within the cell, and the apotransferrin-receptor complex is recycled to the cell surface. Upon encountering the neutral pH of the medium, apotransferrin is dissociated from the cell.