A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [3H]glutamate as radioligand. The rank order of affinity was L-glutamate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) > L(+)-2-amino-3-phosphonopropionic acid (L-AP3) > quisqualic acid > L(+)-2-amino-4-phosphonobutyric acid (L-AP4) > ibotenic acid. The active enantiomers of several phenylglycines displayed Ki values of 300 to 400 microM. The nonactive enantiomers and the standard ionotropic glutamate receptor ligands N-methyl-D-aspartic acid (NMDA), (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid only weakly displaced [3H]glutamate. In this cell line, L-glutamate and (2S,3S,4S)-alpha-(Carboxycyclopropyl)-glycine (L-CCG-I) reduced cAMP levels in a dose-dependent manner. The sensitivity of this system and its easy applicability make it feasible to envisage ligand binding assays on cell lines expressing cloned receptors as useful screening tools to discover and characterize new and specific agonists and antagonists.