Excitatory amino acid transporter 4 (EAAT4), a member of the high-affinity Na+/K+-dependent glutamate transporter family, is highly enriched in Purkinje cells of the cerebellum, although it is not restricted to these cells. The detailed expression of EAAT4 protein in different adult rat fore- and midbrain regions was examined. Despite moderate expression levels compared with the cerebellum, EAAT4 protein was omnipresent throughout the fore- and midbrain. With antibodies raised against the N-terminal mouse EAAT4 sequence, the highest protein expression levels were observed in the substantia nigra pars compacta, ventral tegmental area, paranigral nucleus, habenulo-interpeduncular system, supraoptic nucleus, lateral posterior thalamic nucleus, subiculum, and superficial layers of the superior colliculus. Relatively high levels of EAAT4 protein were also detected in the hippocampal principal cells, in the glutamatergic, gamma-aminobutyric acid (GABA)ergic, dopaminergic and most likely cholinergic cells of all nuclei of the basal ganglia, and in neurons of layers II/III and V of the cerebral cortex. The expression of EAAT4 was confirmed at the mRNA level in some important fore- and midbrain structures by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) and estimated to range from 6.7 to 1.6% of the amount in the cerebellum as measured by real-time PCR.