The glutamate analog (RS)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of l-[ 3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive l-[ 3H]glutamate binding was additive with that displaced by kainic acid (1 μM) plus N-methyl- d-aspartate (10 μM) when low concentrations of non-radioactive AMPA (1 μM) were employed to determine non-specific background, but partially over-lapped when higher concentration of AMPA (100 μM) were used. [ 3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [ 3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0–4 °C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of l-[ 3H]glutamate, and [ 3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with K d values of 28 and 500 nM and B max values of 200 and 1800 fmol/mg protein, respectively. [ 3H]AMPA was inhibited by quisqualate (IC 50 = 60 nM), l-glutamate (2 μM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4- c]- pyridine-7-carboxylic acid (7-HPCA, 5 μM), kainic acid (20 μM) and glutamic acid diethyl ester (21 μM) but insensitive to l-aspartate, ibotenic acid, N-methyl- d-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 μM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [ 3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [ 3HAMPA binding had a similar distribution to that of l-[ 3H]glutamate binding displaced by AMPA (1 μM). However, additiional l-[ 3H]glutamate binding displaceable by 100 μM cold AMPA did not appear to correspond with sites labeled by [ 3H]AMPA and could represent other subpopulations of glutamate binding sites; therefore, concentrations of AMPA of under 10 μM are recommended for displacement of ‘AMPA sites’.