We obtained patch-clamp recordings from neuron-glial cell pairs in cerebellar brain slices to examine the contribution of glutamate (Glu) uptake by Bergmann glial cells to shaping excitatory postsynaptic currents (EPSCs) at the parallel fiber to Purkinje cell synapse. We show that electrical stimulation of parallel fibers not only activates EPSCs in Purkinje cells but also activates inward currents in antigenically identified Bergmann glial cells that invest Purkinje cell synapse with their processes. The inward current is partially due to 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX)- and 2-amino-5-phosphonopentanoic acid (AP5)-sensitive ionotropic Glu receptors, but >/=70% of the current was mediated by D,L-threo-beta-hydroxyaspartate (THA)-sensitive Glu transporters. Glu inward currents were completely and reversibly inhibited by depolarization of Bergmann glial cells to positive membrane potentials allowing biophysical inhibition of Glu uptake into a single glial cell. Inhibition of Glu transport into Bergmann glial cells by voltage-clamping the cell to depolarized potentials caused a reversible increase in spontaneous EPSC frequency in the Purkinje cell. This increase could also be achieved by pharmacological inhibition of Glu transport with the Glu transport inhibitor THA, suggesting that inhibition of Glu uptake into Bergmann glial cells is responsible for the modulation of postsynaptic EPSCs. THA modulation of spontaneous EPSCs could only be observed in the absence of TTX, suggesting primarily a presynaptic effect. Taken together these data suggest that glial Glu uptake can profoundly affect excitatory transmission in the cerebellum, most likely by regulating presynaptic glutamate release.