A cDNA clone for a new metabotropic glutamate receptor, termed mGluR6, was isolated from a rat retinal cDNA library by cross-hybridization with the previously isolated cDNA clone for a metabotropic glutamate receptor. The cloned mGluR6 subtype consists of 871 amino acid residues and exhibits a structural architecture common to the metabotropic receptor family, possessing a large extracellular domain preceding the seven putative membrane-spanning domains. mGluR6 shows the highest sequence similarity to mGluR4 among the metabotropic receptor subtypes and inhibits the forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the cloned cDNA. mGluR6 potently reacts with L-2-amino-4-phosphonobutyrate (L-AP4) and L-serine-O-phosphate, and the potencies of these compounds are one order of magnitude greater than that of L-glutamate. Blot and in situ hybridization analyses indicated that mGluR6 mRNA is restrictedly expressed in the inner nuclear layer of the retina where ON-bipolar cells are distributed. The metabotropic receptor that responds strongly to L-AP4 and L-serine-O-phosphate in ON-bipolar cells is known to mediate glutamate synaptic transmission between photoreceptor cells and ON-bipolar cells. On the basis of the agonist selectivity of mGluR6 and its specific expression in retinal cells, the physiological role of this receptor subtype in the visual system is discussed.