A quantitative autoradiographic assay for a novel L-[3H]glutamate binding site in rodent brain has been developed. Binding to this site was distinguished by its high affinity for quisqualate (QA), ibotenate, glutamate and trans-1-amino-cyclopentyl-1,3-dicarboxylic acid (trans-ACPD), but low affinity for [RS]-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). 'AMPA-insensitive, QA-sensitive [3H]glutamate binding' (AiQsGB) had a heterogeneous distribution in rat brain with high levels observed in molecular layer of cerebellum, striatum, and lateral septum. AiQsGB was reduced in molecular layer of cerebellum in mice lacking Purkinje cells. AiQsGB appears to represent binding to the 'metabotropic' neuronal excitatory amino acid receptor linked to phosphoinositide metabolism.