The current and calcium influx generated by NMDA receptors depend on the concentration of the coagonist glycine, or its analogue d-serine, in the synaptic cleft. If there is no release of glycine, the ionic stoichiometry of the glial GlyT1 glycine transporters expressed near NMDA receptors in the brain should be able to lower the extracellular glycine concentration to below the EC50 for coactivation of NMDA receptors. We examined whether changing the glycine or d-serine concentration in the superfusion solution altered the NMDA receptor mediated component of the synaptic current at the rat cerebellar mossy fibre to granule cell synapse. Adding up to 100 microM glycine or d-serine had no effect, implying that the glycine site is saturated. Using the competitive glycine site antagonist 7-chlorokynurenate, and plausible values for the kinetic parameters of NMDA receptors, we estimate that during activation of the mossy fibres the concentration of glycine or d-serine in the synaptic cleft is at least 4.6 microM or 1.5 microM, respectively, requiring active release of glycine or d-serine.