By a Percoll density-gradient centrifugation of rat hippocampal homogenate, we found a novel subcellular fraction (specific gravity approximately 1.046 g/ml), besides synaptosomes (approximately 1.060 g/ml), which showed a high activity of Na(+)-dependent glutamate uptake. The initial rate of the glutamate uptake in this fraction was as high as twice that in synaptosomes. Activities of choline acetyltransferase and high affinity choline uptake were, on the other hand, much lower. gamma-Aminobutyric acid uptake activity was nearly equivalent in both fractions. Electron microscopic observations revealed that the fraction was morphologically different from synaptosomal or myelin fractions, but mainly consisted of two different types of empty membrane vesicles; irregular (0.3-0.8 micron in diameter) and spheroid type (0.2 micron). The immunoreactivity to glial fibrillary acidic protein was appreciably high in this fraction. The marker enzyme analysis showed the fraction was rich in plasma membranes. On the basis of these results, the fraction is termed glial plasmalemmal vesicles (GPV). We analyzed kinetically the reaction of Na(+)-dependent glutamate uptake by GPV comparing with that by synaptosomes. Km values for glutamate in GPV was 4.7 microM and Vmax was 33 nmol/mg/min, while in synaptosomes 11 microM and 17 nmol/mg/min, respectively. Hill coefficients of Na+ activation in GPV and synaptosomes were 1.1 and 2.0, respectively. Thus, the mechanism or transporter molecule in glial cells for Na(+)-dependent glutamate transport is likely to be different from that in neurons.