The binding of the putative excitatory transmitters glutamate (Glu) and aspartate (Asp) was measured in various subcellular fractions in order to assess their degree of localization in synaptic junctions (SJs). For both ligands, specific binding levels increased in the order, whole particulate membranes--crude mitochondrial pellet membranes (P2)-- synaptic plasma membranes (SPM), and were highly enriched in SJs, with values approximately 9 times greater than the values in whole particulate membranes. The recovery of binding sites in SJs suggested that the majority of sites in SPMs were junctional in nature. Specific binding sites were found also in other subcellular fractions, such as microsomal membranes, “light” SPMs, and mitochondrial membranes. Sodium ions were able to stimulate the specific binding of both ligands (Asp greater than Glu), the magnitude of the effect between subcellular fractions being in the order, whole particulate membranes greater than P2 greater than SPM. This effect was absent in SJs. Calcium and magnesium ions also enhanced the binding (Glu greater than Asp) in the order, whole particulate membranes greater than or equal to P2 greater than or equal to SPM greater than SJ. The results indicate that Glu and Asp binding sites have a specific synaptic localization and support a role for Glu and Asp receptors in synaptic transmission.