In this study, a Müller cell culture preparation from young rats was used to investigate the regulation of GLAST transport activity in native cells. Immunohistochemical analysis confirmed GLAST to be the predominant glutamate transporter expressed by the cells through five passages. [3H]-glutamate uptake assays showed the typical Na+-dependent glutamate transport which was blocked by L-(-)-threo-3-hydroxyaspartate (L-THA), a competitive inhibitor. Glutamate transport was decreased significantly in Müller cells exposed to phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. A similar effect on [3H]-D-aspartate (nonmetabolizable glutamate analog) uptake ruled out the possibility that the decrease was a consequence of altered metabolism. However, PMA did not affect Na+-dependent [3H]-glycine transport, indicating the absence of a nonspecific change in the electrochemical gradients. The PMA effect on glutamate uptake was evidenced by partial blocking with a specific PKC inhibitor, bisindolymaleimide II (Bis II). Activation of PKC did not change the Km, but the Vmax was significantly reduced. Image analysis of Müller cells with biotinylated cell membranes immunolabeled with GLAST shows a reduction of GLAST in the plasma membrane. In conclusion, these data show that rat Müller cells in primary cultures express GLAST and that PKC activation affects GLAST transport activity by decreasing cell surface expression.