Quantitative autoradiography was used to characterize the pharmacological specificity and anatomical distributions of subtypes of L-[3H]glutamate binding sites in rat brain. One population of sites was sensitive to N-methyl-D-aspartate (NMDA) and other compounds thought to be specific for the NMDA receptor. This site was enriched in stratum radiatum of hippocampus (CA1) where it constituted about 80% of glutamate binding sites and it represented a variable portion of glutamate binding sites throughout the brain. A second population of sites had a high affinity for quisqualate. Approximately 80% of glutamate binding sites in cerebellar molecular layer were of the high affinity quisqualate type. The number of these sites was greatly increased in the presence of Cl- and Ca++ ions. A subset of the high affinity quisqualate sites was sensitive to competition by kainate, particularly in stratum lucidum of hippocampus; the density of these high affinity kainate-sensitive sites was decreased in the presence of Ca++ but not Cl- ions. At high concentrations quisqualate competes for all glutamate binding sites, as reported previously. There was a good correspondence between the density and distribution of low affinity quisqualate sites and NMDA-sensitive sites. Pharmacological analysis suggested that the low affinity quisqualate site and the NMDA site are equivalent. Anatomical and pharmacological evidence suggests that the NMDA-, (high affinity) quisqualate- and kainate-sensitive glutamate binding sites may correspond to the physiologically defined NMDA, quisqualate and kainate receptors.