Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. The present study was an investigation of the protein expression and cellular localization of EAAT4 in human and rat brain, and comparison with another neuronal glutamate transporter, EAAT3 (rabbit excitatory amino acid carrier 1; EAAC1). Regional immunoblot analysis of EAAT4, using a monospecific oligopeptide (carboxy-terminal) affinity-purified polyclonal antibody, revealed that the protein was restricted to the central nervous system. The EAAT4 protein was largely expressed in cerebellum, with a much lower expression in hippocampus, neocortex, striatum, brain stem and thalamus. Immunohistochemical studies showed intense EAAT4 immunoreactivity in the human and rat cerebellar Purkinje cells with a somatodendritic localization. Other brain regions including neocortex, hippocampus, striatum showed faint neuropil staining of EAAT4. Immunogold localization identified EAAT4 protein at plasma membranes of Purkinje cell dendrites and spines. In the hippocampus and neocortex, EAAT4 immunoreactivity was found mainly at small calibre dendrites. Rarely, EAAT4 immunoreactivity was found in astrocytic cell processes of forebrain. In the cerebellum, EAAT4 localization partly overlapped with the neuronal localization of EAAT3 (EAAC1). Immunoreactivity for EAAT3 was enriched in the somatodendritic compartment of the Purkinje cells like EAAT4, but EAAT3 was also found in Purkinje cell axons and in boutons in deep cerebellar nuclei, as well as in granular cells and stellate cells. Our results indicate that EAAT4 protein is largely localized to cerebellar cortex and lower levels of EAAT4 protein are present in forebrain by immunoblot and immunohistochemistry. Both neuronal glutamate transporter EAAT3 (EAAC1) and EAAT4 are located at somatodendritic compartment of Purkinje cells, and probably contribute to glutamate re-uptake mechanisms at Purkinje cell synapses.