Metabotropic receptor subtypes have been proposed based on pharmacological, signal transduction and cDNA sequence data. We assessed potential metabotropic binding site subtypes with in vitro quantitative [3H]glutamate autoradiography in adult rat brains in the presence of saturating concentrations of N-methyl-D-aspartate (NMDA) and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). Quisqualate (QUIS) competition curves resolved two differentially distributed binding sites (KIhigh = 17 nM; KIlow = 62 microM). Trans-1-amino-cyclopentane- 1,3-dicarboxylic acid (t-ACPD) and 1S,3R-ACPD displaced [3H]glutamate binding both in the absence and presence of a quisqualate concentration (2.5 microM) that saturates the high affinity sites, suggesting that both sites are linked to metabotropic receptors. We conclude that two metabotropic binding sites with different distributions and pharmacological profiles can be detected with selective [3H]glutamate binding assays.