[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [14C]glutamine accumulated and [14C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na+-independent and unaffected by the Na+-K+-ATPase inhibitor ouabain, whereas glutamate uptake is Na+-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na+ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [14C]glutamate is measured by superfusion technique in order to prevent reuptake, Na+ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca2+) of the [14C]glutamate as well as of endogenous glutamate. The specific activity of the [14C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na+.