Kinetics for uptake and release of glutamate were measured in normal, i.e., nontransformed, astrocytes in cultures obtained from the dissociated, cortex-enriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with an intense uptake following Michaelis-Menten kinetics. The Km (50 micrometer) was reasonably comparable to the corresponding values in brain slices and in other glial preparations. The Vmax (58.8 nmol min-1 mg-1 protein) was, however, much higher than that observed in glial cell lines or peripheral satellite cells, and also considerably higher than that generally reported for brain slices. The release of glutamate was much smaller than the uptake, and only little affected by an increase of the external glutamate concentration, suggesting a net accumulation of glutamate rather than a homoexchange. Such an intense accumulation of glutamate into normal astrocytes may play a major role in brain metabolism and may help keep the extracellular glutamate concentration below excitatory levels.