The sensitivity of the fluorescent dye, 3,3'-diethylthiadicarbocyanine (DiS-C2(5], was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl3 or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na+-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.