Glutamate is the predominant excitatory neurotransmitter in the CNS, and it is removed from the synaptic cleft by sodium-dependent glutamate transport activity. Glutamate transporter-1 (GLT-1) is expressed predominantly in astroglial cells and is responsible for the largest proportion of glutamate transport in the adult forebrain. In the present study, we demonstrate the ability of endogenous and recombinant GLT-1 to form clusters in astrocytic processes and characterize the mobility and physiological importance of these clusters in the regulation of GLT-1 activity in the presence or absence of neurons. At the distal end of C6 glioma cell processes, GLT-1 clusters undergo rapid morphological changes in both shape and size, and these changes are inhibited by cytochalasin D treatment, suggesting that the morphogenesis of GLT-1 clusters is highly dependent on the actin network. Treatment of astrocytes with phorbol 12-myristate 13-acetate (PMA) quickly and preferentially decreases GLT-1 localization on the process membrane, leading tode novogeneration of GLT-1 clusters along the process shaft. Pretreatment with the PKC inhibitor bisindolylmaleimide II (Bis II), with sucrose (0.4m), or through the expression of a dominant-negative form of dynamin prevents PMA-induced GLT-1 internalization and cluster formation. In terms of glutamate transporter function, PMA treatment elicits a significant decrease in GLT-1 activity that is prevented by preexposure to either Bis II or hypertonic treatment. Together, these data indicate that GLT-1 trafficking and cluster formation in glial cell processes are dynamic events that play important roles in regulating glutamate uptake in astrocytes and glioma cells.