Whole-cell recordings of amacrine and ganglion cells in the superfused retina-eyecup preparation of the mudpuppy were obtained in order to determine which excitatory amino acid receptor (EAAR) subtype mediates the synaptic responses of these neurons. All third-order retinal neurons tested were depolarized by kainic acid (KA, N-methyl-D-aspartate (NMDA), and quisqualate (QQ). The responses evoked by NMDA were blocked by the addition of D-2-amino-5-phosphonovaleric acid (D-AP5) and D-2-amino-7 phosphonoheptonoic acid (D-AP7) to the perfusate. When the actions of exogenously applied NMDA were completely blocked by D-AP5 and D-AP7, the light-evoked responses of inner retinal neurons persisted without any apparent reduction or, alternatively, a slight enhancement of the response was observed. Light-evoked responses of bipolar, amacrine, and ganglion cells associated with the On pathway were attenuated by L-AP5 in a manner similar to its lower-order homolog L-2-amino-4-phosphonobutyrate (AP4); nevertheless, L-AP5 was not an effective NMDA antagonist. Although synaptic transmission between retinal second- and third-order neurons appears to be mediated by EAARs, the NMDA receptor does not appear to play a prominent role under our experimental conditions. Nevertheless, our results suggest that the racemic mixture of AP5 should not be used as an NMDA antagonist in retinal research, due to the AP4-like actions of its L-enantiomer.