We have combined electrophysiology and chemical separation and measurement techniques with capillary electrophoresis (CE) to evaluate the role of endogenous D-serine as an NMDA receptor (NMDAR) coagonist in the salamander retina. Electrophysiological experiments were carried out using whole cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative response (PNR), while bath applying two D-serine degrading enzymes, including Damino acid oxidase (DAAO) and D-serine deaminase (DsdA). The addition of either enzyme resulted in a significant and rapid decline in the light-evoked responses observed in ganglion cell and PNR recordings. The addition of exogenous D-serine in the presence of the enzymes restored the light-evoked responses to the control or supracontrol amplitudes. Heat-inactivated enzymes had no effect on the light responses and blocking NMDARs with AP7 eliminated the suppressive influence of the enzymes as well as the response enhancement normally associated with exogenous D-serine application. CE was used to separate amino acid racemates and to study the selectivity of DAAO and DsdA against D-serine and glycine. Both enzymes showed high selectivity for D-serine without significant effects on glycine. Our results strongly support the concept that endogenous D-serine plays an essential role as a coagonist for NMDARs, allowing them to contribute to the light-evoked responses of retinal ganglion cells. Furthermore under our experimental conditions, these coagonist sites are not saturated so that modulation of NMDAR sensitivity can be achieved with further modulaton of D-serine.