Fluorescence changes of rhodamine 6G in synaptosomal suspension, which are correlated to changes in membrane potential in synaptosomes, were measured in the presence of various monosaccharides and organic acids. Addition of d-glucose, d-mannose, pyruvate and l-lactate hyperpolarized the membrane potential, whereas d-fructose, l-glucose, d-galactose, citrate, succinate and l-glutamate were without effect on the membrane potential. Hyperpolarization induced by d-glucose was inhibited by cytochalasin B, phloretin, iodoacetate, F − and 2-deoxy- d-glucose, but not inhibited by oligomycin or phlorizin. On the other hand, hyperpolarization induced by pyruvate was inhibited by α-cyanocinnamate or phloretin, but not inhibited by cytochalasin B or F −. Elimination of Na + in physiological saline depressed hyperpolarization of membrane potential induced by addition of d-glucose, l-lactate or pyruvate. These results suggest that the activity of (Na + + K +)-ATPase in plasma membranes of synaptosomes is increased by ATP formed by glycolysis, and that the accumulated K + in synaptosomes hyperpolarizes the membrane potential.