[3H]-N-Acetylaspartylglutamate (NAAG) bound saturably and reversibly to crude synaptosomal rat brain membranes. Optimal binding occurred in Tris-HCl buffer, pH 7.2, at 37 degrees C using previously frozen, preincubated membranes. Saturation experiments revealed an apparent KD of 383 +/- 33 nM and a Bmax of 31 +/- 2 pmol/mg of protein. [3H]NAAG specific binding was displaceable by serine-o-sulfate, quisqualate, ibotenate, and glutamate with K1's in the nanomolar range, whereas the amino-phosphono analogues displaced [3H] NAAG in the micromolar range (APB greater than APV greater than APH). No specific binding was found in peripheral tissues. Within the central nervous system, the thalamus exhibited the greatest amount of binding, whereas binding was lowest in cortex. Calcium ions enhanced the specific binding, whereas sodium ions caused a concentration-dependent inhibition. These results suggest that [3H]NAAG labels an acidic amino acid receptor site designated “A-4,” which recognizes the antagonist, 2-amino-4-phosphonobutyric acid, and that this receptor may mediate the neurophysiologic effects of endogenous NAAG.