Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. J. Neurophysiol. 80: 3336-3340, 1998. At central synapses occupation of glycine binding sites of N-methyl--aspartate receptors (NMDA-Rs) is a necessary prerequisite for the excitatory neurotransmitter glutamate to activate these receptors. There is conflicting evidence as to whether glycine binding sites normally are saturated. If they are not, then alterations in local glycine concentration could modulate excitatory synaptic transmission. By using an in vitro brain stem slice preparation we investigated whether the glycine site is saturated for synaptically activated NMDA-Rs in neonatal rat hypoglossal motoneurons. We found that the NMDA-R-mediated component of spontaneous miniature excitatory postsynaptic currents could be potentiated by exogenously applied glycine as well as by -serine. The effects of glycine were observed only at concentrations (100 microM or more) two orders of magnitude above the apparent dissociation constant of glycine from NMDA receptors. In contrast, -serine, a nontransported NMDA-R glycine site agonist, was effective in the low micromolar range, i.e., at concentrations similar to those found to be effective on isolated cells or on outside-out patches. We conclude that at these synapses the glycine concentration around synaptic NMDA-Rs is set below the concentration required to saturate their glycine site and is likely to be stabilized by a powerful glycine transport mechanism.