Glutamate is a key neurotransmitter and its levels in the synaptic cleft are tightly regulated by reuptake mechanisms that primarily involve transporters in astrocytes. This requires that the glutamate transporters be spatially constrained to effect maximum glutamate transport. GLAST (EAAT1) is the predominant astroglial transporter and contains a class I PDZ-binding consensus (ETKM) in its C-terminus. The epithelial Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2 are PDZ proteins that contain two tandem PDZ domains and a C-terminal domain that binds members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adaptors. NHERF proteins have been extensively characterized in renal epithelia and their expression in brain has recently been reported; however, their function in the brain remains unknown. The aims of the current study were to (1) determine the distribution of NHERF1/2 in the rodent brain and (2) investigate whether GLAST was a physiological ligand for NHERF1/2. Immunohistochemistry revealed that NHERF1 expression was widespread in rat brain (abundant in cerebellum, cerebral cortex, hippocampus, and thalamus) and primarily restricted to astrocytes whereas NHERF2 expression was primarily restricted to endothelial cells of blood vessels and capillaries. Importantly, NHERF1 distribution closely matched that of GLAST and confocal imaging demonstrated co-localization of the two proteins. Co-immunoprecipitation demonstrated that GLAST, NHERF1, and ezrin associate in vivo. In vitro binding assays showed that GLAST bound directly to the PDZ1 domain of NHERF1 via the C-terminal ETKM motif of GLAST. These findings implicate the GLAST-NHERF1 complex in the regulation of glutamate homeostasis in astrocytes.