d-serine occurs at high levels in the brain, where it is an endogenous coagonist at the “glycine site” of NMDA receptors. However, d-serine action has not been previously compared with that of endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous d-serine from slices was achieved by pretreating the tissue with recombinant d-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove d-serine. We report that complete removal of d-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than d-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous d-serine and blockage of the glycine transporter GlyT1. Our data indicate that d-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices. The results suggest an essential role for this unusual d-amino acid, with implications for the mechanism of neuronal death in the nervous system.