Quantitative autoradiographic techniques were used to obtain a preliminary description of the pharmacological characteristics and anatomical distribution of the selective N-methyl-D-aspartate (NMDA) receptor antagonist D-2-[3H]amino-5-phosphonopentanoate (D-[3H]AP5). Binding sites exhibit the appropriate pharmacological profile and are found throughout the brain. Consistent with ligand binding experiments using purified synaptic membranes, the hippocampus has the highest levels of binding. Within this structure, the binding site distribution is indistinguishable from that obtained for D-AP5-sensitive L-[3H]glutamate binding sites; highest levels are found within the stratum radiatum, a region in which NMDA receptors are involved in the formation of long-term potentiation.