Iron uptake by rat reticulocytes is blocked by 20 mM NH4Cl, while 125I-diferric transferrin (Tf) uptake is relatively unaffected. At pH 5.0 both apo- and diferric Tf bind with high affinity; at pH 7.4 diferric Tf binds avidly, but apoTf binds very poorly. The dissociation rate (4 degrees C) of diferric Tf is extraordinarily slow at pH 5.0 (extrapolated t 1/2 = 32 hrs) and faster at pH 7.4 (t 1/2 = 101 min). At pH 5.0 apoTf also dissociates slowly (t 1/2 = 205 min), but at pH 7.4 apoTf exhibits a much faster dissociation rate (t 1/2 = 62 min). 20 mM NH4Cl slows the release of Tf from cells at 37 degrees C, but the rate of externalization of ligand is unaffected. Ligand dissociation at 37 degrees involves both externalization of receptor-ligand complexes and receptor-ligand separation; the NH4Cl effect may result from an increased fraction of externalized Tf in the diferric form which may dissociate more slowly. Receptor-mediated movement of Tf through acid intracellular compartments provides a mechanism to remove iron from Tf and for apoTf to remain receptor-bound for externalization to the cell surface and subsequent dissociation.