The effect of excitatory amino acids on the uptake of 45Ca was studied in crude mitochondrial (P2) fractions prepared from mouse brain. L-Glutamate stimulated calcium uptake, but this action was not shared by other amino acids including D-glutamate, L-aspartate, N-methyl-aspartate or kainate. The glutamate-stimulated calcium uptake was, however, blocked by inhibitors of glutamate dehydrogenase, such as D-glutamate, glutarate and triiodothyronine. Subcellular fractionation demonstrated that the uptake was enriched in the mitochondrial fraction. Furthermore, most of the uptake found in the synaptosomal fraction was inhibited by triiodothyronine. These results indicate that the glutamate-stimulated uptake of calcium by brain membranes is due mainly to mitochondrial uptake of calcium that is driven by the metabolism of glutamate by glutamate dehydrogenase. Previous suggestions of coupling of glutamate receptors to calcium channels based on uptake of 45Ca by brain membranes must now be reevaluated.