Membrane vesicles, isolated after osmotic shock of synaptosomal rat brain fractions, actively accumulate L-glutamate. This process requires the presence of external sodium ions and internal potassium ions and is driven by artifically imposed ion gradients as the sole energy source. Either an Na+ gradient (out is greater than in) or a K+ gradient (in is greater than out) or both can be utilized to concentrate L-glutamate inside the vesicles. Transport is enhanced by valinomycin or by external thiocyanate ions and is about 50% inhibited by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. This transport thus appears to be stimulated by a membrane potential (interior negative). The glutamate transporter, the Km of which has been determined to be 3 micrometer, is specific for L-glutamate. The transport process is unaffected by ouabain but is strongly inhibited by p-hydroxymercuribenzoate as well as by nigericin, which collapses the energizing ion gradients across this membrane. Unlike the sodium dependent, but potassium independent active accumulation of gamma-aminobutyric acid in these vesicles (Kanner, B.I. (1978) Biochemistry 17, 1207) active L-glutamate uptake is not dependent on the presence of small monovalent anions in the external medium. The results provide direct evidence for Na+-coupled electrogenic active L-glutamate transport by rat brain membrane vesicles. The dependence on internal potassium ions is discussed.