Chronic exposure to cadmium (Cd) results in an inhibition of protein endocytosis in the renal proximal tubule, leading to proteinuria. In order to gain insight into the mechanism by which Cd impairs the protein endocytosis, we investigated the effect of Cd on the acidification of renal cortical endocytotic vesicles (endosomes). The endosomal acidification was assessed by measuring the pH gradient-dependent fluorescence change, using acridine orange or FITC-dextran as a probe. In renal endosomes isolated from Cd-intoxicated rats, the Vmax of ATP-driven fluorescence quenching (H⁢-ATPase dependent intravesicular acidification) was significantly attenuated with no substantial changes in the apparent Km, indicating that the capacity of acidification was reduced. When endosomes from normal animals were directly exposed to free Cd in vitro, the Vmax was slightly reduced, whereas the Km was markedly increased, implying that the biochemical property of the H⁢-ATPase was altered by Cd. In endosomes exposed to free Cd in vitro, the rate of dissipation of the transmembrane pH gradient after H⁢-ATPase inhibition appeared to be significantly faster compared to that in normal endosomes, indicating that the H⁢-conductance of the membrane was increased by Cd. These results suggest that in long-term Cd-exposed animals, free Cd ions liberated in the proximal tubular cytoplasm by lysosomal degradation of cadmium-metallothionein complex (CdMT) may impair endosomal acidification 1) by reducing the H⁢-ATPase density in the endosomal membrane, 2) by suppressing the intrinsic H⁢-ATPase activity, and 3) possibly by increasing the membrane conductance to H⁢ ion. Such effects of Cd could be responsible for the alterations of proximal tubular endocytotic activities, protein reabsorption and various transporter distributions observed in Cd-exposed cells and animals.