The uptake of L-glutamate was studied in astrocytes cultured from different brain areas of newborn rats as well as in two different cultures of neurons obtained from mouse brain. Both astrocytes and neurons exhibited high-affinity glutamate uptake with Km values ranging from 34 microM to 82 microM. Vmax values for astrocytes cultured from the different brain regions were: prefrontal cortex: 13.9; occipital cortex: 11.4; neostriatum: 27.3 and cerebellum: 5.8 nmol X min-1 X mg-1 cell protein. For cerebellar granule cells and cerebral cortical neurons the Vmax values were found to be 10.2 and 5.9 nmol X min-1 X mg-1 cell protein, respectively. The effect on L-glutamate uptake in astrocytes cultured from prefrontal cortex and in cultured cerebellar granule cells of a series of compounds structurally related to glutamate was studied, and detailed kinetic analyses of the inhibitory patterns of three potent inhibitors were performed. L-aspartate and L-aspartate-beta-hydroxamate were found to be competitive inhibitors of L-glutamate uptake in both cell types with Ki values for astrocytes of 60 microM and 91 microM, respectively, and for granule cells of 48 microM and 72 microM, respectively. D-aspartate was found to be a mixed-type noncompetitive inhibitor of L-glutamate uptake in astrocytes (Ki: 106 microM), but in granule cells this compound showed simple competitive inhibition with a Ki of 49 microM. Sodium dependency of L-glutamate uptake in both cell types was studied at a series of L-glutamate and Na+ concentrations. It was found that the uptake of glutamate in astrocytes is coupled with one Na+ ion in contrast to two Na+ ions in granule cells. The Km value for sodium was found to be 15 mM in both cell types. It was shown that release of exogenously supplied [3H]-L-glutamate from cerebellar granule cells could be stimulated in a Ca2+-dependent manner by high concentrations (55 mM) of K+. In contrast to this no K+-induced release of glutamate could be demonstrated in cultured astrocytes.