The Na+,glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (Iglu). Coexpression of either, SGK1 or PIKfyve, significantly enhanced Iglu in EAAT2 expressing oocytes. Iglu was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase K127NSGK1 did not significantly alter Iglu in EAAT2 expressing oocytes and significantly decreased Iglu in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on Iglu was abrogated by replacement of the serine in the SGK consensus sequence by alanine (S318APIKfyve). Furthermore, additional coexpression of S318APIKfyve virtually abolished Iglu in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed articipates in the regulation of EAAT2.