Glutamate is thought to serve as a major excitatory neurotransmitter throughout the central nervous system (CNS); electrophysiological studies indicate that its action is mediated by multiple receptors. Four receptors have been characterized by their selective sensitivity to N-methyl-D-aspartate (NMDA), kainic acid (KA), quisqualic acid (QA) and 2-amino-4-phosphonobutyric acid (APB). Electrophysiological evidence indicates that these receptors are all present in the rat hippocampus and that the anatomically discrete synaptic fields within the hippocampus exhibit differential sensitivity to the selective excitatory amino acid agents. Thus, we have used the hippocampus as a model system to investigate possible subpopulations of 3H-L-glutamate binding sites. By using quantitative autoradiography, the pharmacological specificity of 3H-L-glutamate binding in discrete terminal fields was determined. We report here that there are at least four distinct classes of 3H-L-glutamate binding sites which differ in their anatomical distribution, pharmacological profile and regulation by ions. Two of these sites seem to correspond to the KA and NMDA receptor classes, and a third site may represent the QA receptor. The fourth binding site does not conform to present receptor classifications. None of these binding sites corresponds to the major glutamate binding site observed in biochemical studies.