The gene for EAAT2, the major astrocytic glutamate transporter, generates two carrier isoforms (EAAT2a and EAAT2b) that vary at their C termini as a consequence of alternative RNA splicing. The EAAT2b cytoplasmic C terminus contains a postsynaptic density-95/Discs large/zona occludens-1 (PDZ) ligand, which is absent in EAAT2a. To understand how the distinct C termini might affect transporter trafficking and surface localization, we generated Madin-Darby canine kidney (MDCK) cells that stably express EGFP-EAAT2a or EGFP-EAAT2b and found robust basolateral membrane expression of the EAAT2b isoform. In contrast, EAAT2a displayed a predominant distribution within intracellular vesicle compartments, constitutively cycling to and from the membrane. Addition of the PDZ ligand to EAAT2a as well as its deletion from EAAT2b confirmed the importance of the motif for cell-surface localization. Using EAAT2 constructs with an extracellular biotin acceptor tag to directly assess surface proteins, we observed significant PDZ ligand-dependent EAAT2b surface expression in cultured astrocytes, consistent with observations in cell lines. Discs large homolog 1 (DLG1; SAP97), a PDZ protein prominent in both astrocytes and MDCK cells, colocalized and coimmunoprecipitated with EAAT2b. shRNA knockdown of DLG1 expression decreased surface EAAT2b in both MDCK cells and cultured astrocytes, suggesting that the DLG scaffolding protein stabilizes EAAT2b at the surface. DLG1 can be phosphorylated by Ca(2+)/calmodulin-dependent protein kinase (CaMKII), resulting in disruption of its PDZ-mediated interaction. In murine astrocytes and acute brain slices, activation of CaMKII decreases EAAT2b surface expression but does not alter the distribution of EAAT2a. These data indicate that the surface expression and function of EAAT2b can be rapidly modulated through the disruption of its interaction with DLG1 by CaMKII activation.