Potassium chloride permeability and relative ionic conductances of rat renal cortical brush-border (BBMV) and basolateral (BLMV) membrane vesicles were examined using the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. Vesicles were simultaneously isolated and separated by free-flow electrophoresis. These studies demonstrated that neither BBMV nor BLMV equilibrated in 100 mM KCl despite prolonged incubation. In both, an inwardly directed KCl gradient was sustained for 3 h. The low intravesicular KCl concentration of BLMV was confirmed utilizing the response of electrogenic Na+-dependent [3H]glutamine transport to variations in the membrane potential. Chloride conductance was significantly less than potassium conductance in BBMV and BLMV. Consequently, an inside-positive potential was maintained across both membranes. BLMV were significantly more fluid, less permeable, had a lower relative chloride conductance, and maintained a greater inside-positive potential than BBMV. The KCl permeabilities of BBMV and BLMV were inversely related to endogenous membrane copper content and were significantly reduced by exogenous copper. Permeability did not correlate with membrane magnesium content, nor was it affected by exogenous magnesium. These studies suggest that endogenous copper as well as intracellular factors may regulate the permeabilities of the brush-border and basolateral membranes of proximal tubule cells in vivo.