L-[3H]Proline binds specifically and saturably to rat hippocampal synaptic membranes in the absence of inorganic ions. Saturation isotherms constructed in Hepes-Tris buffer best fit a model of a single independent population of binding sites with a dissociation constant of 1.6 microM and maximum density of 16 pmol/mg protein. The rank order of displacement of radioligand by proline analogues suggests that binding sites of this type could be receptors responsible for proline-induced retinal spreading depression and inhibition of memory consolidation.