Abstract The role of glutamate as a possible mediator of neurodegeneration is well described, and the homeostasis of extracellular glutamate is considered of major importance when addressing the pathogenesis of excitatory neurodegeneration. Applying the ‘indicator diffusion’ method to the microdialysis technique, we present a method that is suitable for the in vivo investigation of the capacity of cellular uptake of glutamate. Using 14C-mannitol as reference, we measured the cellular extraction and the cell membrane permeability of the test substance 3H- d-aspartate in the corpus striatum of the rat brain. The cellular extraction fraction of 3H- d-aspartate was 0.29, and the cell membrane permeability 2.24 × 10 −4 cm/s. In the presence of the glutamate-uptake blocker dl-threo-β-hydroxyaspartate (THA) the extraction of 3H- d-aspartate was completely abolished, indicating that extraction of 3H- d-aspartate was due to cellular uptake by glutamate transporters. The cell membrane permeability towards 3H- d-aspartate was reduced by ≈ 98% due to THA, indicating that the cell membranes per se are highly resistant to diffusion of 3H- d-aspartate. It is concluded that the present method can be used in studying the capacity of the glutamate transporters in vivo.