Metabotropic glutamate receptors are selectively activated by 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). [3H]Glutamate binding sites in rat brain membranes were characterized in the presence of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) to block binding to ionotropic glutamate receptors. 1S,3R-ACPD displaced a single population of [3H]glutamate binding sites and was mimicked by other metabotropic glutamate agonists with a potency order of L-glutamate > 1S,3R-ACPD > ibotenate > 1R,3S-ACPD. Quisqualate interacted at two populations of binding sites. 1S,3R-ACPD-sensitive [3H]glutamate binding was saturable (Bmax = 2.50 +/- 0.27 pmol/mg protein), reversible, and had high-affinity (KD = 187 +/- 60 nM). 1S,3R-ACPD-sensitive [3H]glutamate binding likely represents labeling of metabotropic glutamate receptors in rat brain membranes.