Uptake of host transferrin (Tf) in Trypanosoma brucei is mediated by a heterodimeric, glycosyl-phosphatidylinositol-anchored receptor. After endocytosis, Tf is delivered to lysosomes where it is proteolytically degraded. So far, the sequence of events leading to ligand dissociation and degradation is undefined. We now show by Triton X-114 phase separation that iron-free Tf (apo-Tf) dissociates from the receptor at pH 5.0. The low affinity of apo-Tf for its receptor at pH 5.0 is confirmed by an apparent dissociation constant of 1.1 microM. The implications of this result on the mechanism of intracellular processing of Tf in trypanosomes are discussed.