Glycine and the divalent cation Ca2+ play key roles in regulating the activity of excitatory amino acid NMDA receptor channels. There is accumulating evidence that the concentration of glycine at the synaptic cleft is below a saturated level. We examined the effect of external Ca2+ on NMDA responses in various concentrations of glycine in isolated trigeminal neurons. We found that external Ca2+ potentiated NMDA responses and this potentiation occurred only when glycine sites were unsaturated. Since single-channel conductance decreases in the high external Ca2+ solution, the observation cannot be explained by an increase in Ca2+ influx through the channels. Studying the dose- response curves for glycine in different Ca2+ solutions, we found that the apparent dissociation constant (EC50) for glycine decreases with increasing external Ca2+ concentrations. Kinetics studies of glycine binding to NMDA receptors indicated that external Ca2+ causes a decrease in the off rate of the glycine binding, while having no effect on the on rate. Our analyses suggest that the apparent glycine affinity increases by about 3.7 times in Ca-containing solution. Thus, external Ca2+ contributes to the unusually high glycine affinity for NMDA receptors and may have a role in regulating the NMDA receptor channel activities during intensive or sustained neuronal stimulation.