The actions of glycine on the NMDA receptor-mediated synaptic responses of ganglion cells were studied in the tiger salamander retinal slice. Ganglion cell excitatory postsynaptic currents (EPSCs) were elicited either by exciting bipolar cells with potassium puffs or by light stimulation, and were measured using whole-cell patch-clamp techniques. Increasing bath glycine concentrations to 10 microM had little effect on the amplitude of the puff-evoked EPSCs, indicating either that synaptic glycine concentrations were saturating or that the added glycine was buffered by uptake mechanisms. However, 5,7-dichlorokynurenic acid (5,7-DCK), an antagonist for the glycine site on the NMDA receptor, reduced the ganglion cell responses to NMDA puffs, and reduced the potassium puff- and light-elicited EPSCs. The IC50 values for 5,7-DCK became larger with increasing glycine concentrations, but not with increasing NMDA concentrations, indicating that 5,7-DCK acted at the glycine site. The IC50 values for 5,7-DCK were increased with stronger potassium puffs or light stimuli, suggesting that synaptic glycine levels increased with the strength of the stimuli. EPSCs measured in ON-OFF ganglion cells at light ON and OFF were reduced by 5,7-DCK. For dim light stimuli, the IC50 values were lower for the OFF EPSCs compared to the ON EPSCs, indicating that glycine concentrations were different at the times of OFF and ON activity. Estimates of synaptic glycine concentrations suggest that for dim light stimuli, concentrations at the OFF synapses were not saturated, but concentrations at the ON synapses were saturated.