Recently, we isolated a novel subcellular fraction of glial plasmalemmal vesicles (GPV), which showed a higher activity of Na(+)-dependent glutamate transport than synaptosomes (Nakamura et al., 1993). In order to study kinetically the glutamate transport mechanism, we measured the reaction under various ionic conditions both inside and outside the vesicles. The vesicles treated hypotonically and preloaded with KCl could take up glutamate in the presence of external Na+. The level of glutamate uptake was dependent on external concentrations of NaCl ([NaCl]o) and competitively inhibited by [KCl]o. However, it was dependent on [KCl]i, and competitively inhibited by [NaCl]i. The activation and inhibition constants of K+ were about 30 mM inside and 20 mM outside, respectively, whereas those of Na+ were 140 mM outside and 4 mM inside, respectively. These results suggest that the transport carrier molecules work asymmetrically to the membranes. Nigericin and monensin, acidic ionophores for K+ and Na+, respectively, inhibited the glutamate uptake. On the other hand, valinomycin, a neutral ionophore for K+, elevated the uptake level, suggesting that the inside-negative membrane potential induced by K+ diffusion enhances the uptake activity. We conclude that glutamate transport by glial cells requires both external Na+ and internal K+ and is regulated by the membrane potential.