Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC50=44.7±12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4α-phorbol-12,13-didecanoate (4α-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in Imax with no change in Km. PMA simultaneously decreased membrane capacitance (Cm) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific. © 2001 Elsevier Science B.V. All rights reserved.