The metabolism of [1-13C]D-glucose by astrocytes, neurons and mixed astroglial/neuronal cultures derived from the striatum of fetal rats was studied using NMR. Metabolic activity was studied in resting and depolarized cells (55 mM K+), with dibutyryl cyclic-AMP added to the medium to promote cell differentiation, and with glutamate (0.1 mM) included in the medium. Due to sample limitations the accumulation of 13C label in metabolites within the cells was not sufficient to quantitate. Of the metabolites released into the medium by the astrocyte cultures and the mixed astroglial/neuronal cultures, measurable amounts of label were present in lactate C-3 and C-2, glutamine C-2, C-3 and C-4, acetate C-2, citrate C-2 or C-4 and C-3, glycerol C-1 or C-3, succinate C-2 or C-3 and several unidentified metabolites. Of the labeled metabolites released into the medium, only succinate was markedly affected by K(+)-induced depolarization, dBcAMP, or glutamate. The label in succinate was increased, especially in the K(+)-depolarized astrocyte cultures (3- to 6-fold). The neuronal cultures consumed [1-13C]D-glucose much more slowly than the astrocyte cultures or the mixed cultures. Except for lactate C-3, there was no measurable 13C in metabolites in the medium of the neuronal cultures.