NMDA receptors are ligand-gated cation-selective channels that mediate a slow, Ca2+-permeable component of the excitatory postsynaptic current. These receptors are involved in a wide range of important functions in the central nervous system, including synaptic plasticity and development. In addition, these receptors have been implicated in several neurological diseases, including depression, schizophrenia, Parkinson’s disease, and epilepsy. We have, in collaboration with the NIH Undiagnosed Disease Program, investigated de novo mutations that have been found to arise in patients with varying neurological disorders, including seizures or developmental delay. Next generation high throughput whole exome sequencing of patients, parents, and relatives together with unique de novo analysis routines has identified a single candidate mutation within NMDA receptor subunits GRIN2A and GRIN2B for each patient. These mutations were subsequently introduced into recombinant human NMDA receptor cDNAs encoding the GluN2A and GluN2B subunits, which were studied in heterologous expressions systems using voltage clamp recordings to assess their effects on channel function. We utilized two electrode voltage recordings from Xenopus laevis oocytes injected with cRNA transcribed in vitro to study pharmacological properties of mutant human GluN1/GluN2 NMDA receptors. These experiments were complemented with patch clamp recordings in HEK 293 cells transfected with cDNA encoding the human GluN1/GluN2 NMDA receptor subunits. The goal of these functional studies was to determine whether the changes in receptor properties produced by these mutations were potentially disease-causing. In both cases the mutations had strong functional effects that were highly suggestive of these mutations being disease-causing. The location of the mutation implicated a key residue in the glutamate binding domain of GluN2B and S2-M4 GluN2A linker. The loss of function GluN2B mutation showed dramatically lower sensitivity to glutamate (50-fold higher EC50) without a change in glycine potency, and was associated clinically with severe developmental delay. The GluN2A gain-of-function mutation increased both glutamate and glycine potency 8-fold and increased open probability, leading to hyperactive NMDA receptors. The overactivation of NMDA receptors may account for frequent seizures. Experiments with memantine showed that it remained an effective antagonist against mutant NMDA receptors, suggesting a therapeutic course of action, which when implemented improved the patient’s condition. We are currently expanding our search for potential disease causing mutations in the glutamate receptor family. Identification and characterization of such mutations provides an opportunity both to better understand glutamate receptor function, as well as the role of glutamate receptors in human disease.