Antibodies to an excitatory amino acid transporter (EAAT4) label a glycoprotein of approximately 65 kDa strongly in the cerebellum and weakly in the forebrain. Cross-linking of cerebellar proteins with bis(sulfosuccinimidyl) suberate before solubilization causes dimer bands of EAAT4 and both dimer and trimer bands of the other glutamate transporters GLAST (EAAT1) and GLT (EAAT2) to appear on immunoblots. In contrast to GLAST, GLT, and EAAC (EAAT3), EAAT4 is unevenly distributed in the cerebellar molecular layer, being strongly expressed in parasagittal zones. It is located in cerebellar Purkinje cells, and the highest concentrations are seen in ones expressing high levels of zebrin II (aldolase C). The labeling of Purkinje cell spines and thin dendrites is stronger than that of large diameter dendrites and cell bodies. EAAT4 is present at low concentrations in the synaptic membrane, but is highly enriched in the parts of the dendritic and spine membranes facing astrocytes (which express GLAST and GLT) compared with parts facing neuronal membranes, suggesting a functional relationship with glial glutamate transporters. The presence of EAAT4 in intracellular cisterns and multivesicular organelles may reflect turnover of transporter in the plasma membrane. The total Purkinje cell spine surface and the EAAT4 concentration were found to be 1.1 m2/cm3 and 0.2 mg/cm3, respectively, in the molecular layer, corresponding to 1800 molecules/microm2. The juxtasynaptic location of EAAT4 may explain electrophysiological observations predicting the presence of a neuronal glutamate transporter near the release site at a Purkinje cell spine synapse. EAAT4 may function as a combined transporter and inhibitory glutamate receptor.