NMDA receptor (NMDAR) mediated spontaneous miniature excitatory postsynaptic currents (mEPSCs) are potentiated by exogenously applied glycine (Berger et al. 1998). In this study we have investigated the affect of blocking glycine uptake on NMDAR-mediated responses from hypoglossal motorneurons (HMs) of rats. We have used N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine (NFPS; 500nM) an antagonist of glycine transporter-1 (GLYT1) to study the effect of blocking endogenous glycine uptake on NMDAR-mediated synaptic transmission. We show that the charge transfer of NMDAR-mediated mEPSCs was enhanced following NFPS application in neonate (P2-4) and juvenile rats (P8-11), but this enhancement was statistically significant only in the former group. Spontaneous EPSCs and evoked EPSCs showed a significant increase in NMDAR-mediated charge transfer in both neonates and juveniles. The greater increase observed in sEPSCs may be due to increased release of glycine from glycinergic terminals in the absence of tetrodotoxin (TTX). Brief application of NMDA onto HMs showed that extrasynaptic NMDARs may be potentiated by NFPS only in the presence of extracellularly applied glycine. Immunohistochemistry of GLYT1 and 2 shows labeling throughout the hypoglossal nucleus. GLYT1 labeling is diffuse and becomes more intense and uniform during development consistent with its glial localization. In contrast, GLYT2 labeling is intense throughout the nucleus and increases in intensity with age. Our results demonstrate the glycine binding site of the NMDAR is not saturated in the brainstem slice during the first two weeks of development. We suggest that modulation of glycine concentration by GLYT1 is an important mechanism to regulate NMDAR-mediated synaptic transmission.