The excitatory amino acid receptors selectively activated by N-methyl-D-aspartate (N-Me-D-Asp) (also known as NMDA) are a major determinant of central nervous system neuronal excitability. We report here that rat brain synaptic plasma membranes contain a distinct population of L-[3H]glutamate binding sites with pharmacological properties indicative of the N-Me-D-Asp receptor. The N-Me-D-Asp sites are readily distinguished from other L-[3H]glutamate binding and uptake sites by their sharp pH optimum, more rapid association rate, preferential localization in synaptic structures, and lack of dependence on temperature and inorganic ions. As with other receptor systems, ligand binding at the N-Me-D-Asp site is reduced by guanine nucleotides but not by adenosine nucleotides. Binding is insensitive to ketamine and cyclazocine, indicating that sigma opiates inhibit N-Me-D-Asp excitation at a site different from that of the N-Me-D-Asp binding site. The quantitative pharmacological properties of N-Me-D-Asp-sensitive L-[3H]glutamate binding sites determined in a well-defined dendritic field (stratum radiatum of CA1) by quantitative autoradiography closely correlate to those of both the electrophysiologically identified N-Me-D-Asp receptors in the same dendritic field and the N-Me-D-Asp sites studied in membrane preparations. Under conditions that selectively reveal N-Me-D-Asp receptors, these sites are found to exhibit considerable anatomical specificity as evidenced by variations within cortical, striatal, and thalamic regions. Autoradiography also showed that regions in rodent and primate brain that are especially sensitive to anoxic and excitotoxic neuronal damage (e.g., Sommer's sector or CA1) have a high level of N-Me-D-Asp sites. Since N-Me-D-Asp receptors are known to contribute to these causes of neuronal loss, their selective distribution partially accounts for the pattern of selective damage seen in these pathological conditions.