The cDNA encoding the human metabotropic glutamate receptor type 6 (hmGlu6) was isolated from a human retinal cDNA library. The deduced primary sequence (877 amino acids) of the hmGlu6 receptor was 93.5% identical to its rat counterpart and shared 69.8% sequence identity with the related hmGlu4 receptor clone (912 amino acids), isolated in parallel from a human brain cDNA library. In situ hybridization revealed that the hmGlu6 mRNA is highly expressed in cells located in the inner nuclear layer of the human retina, presumably bipolar neurons. Neither PCR analysis nor in situ hybridization could detect hmGlu6 mRNA in human brain. When stably expressed in Chinese hamster ovary cells (CHO-K1) the hmGlu6 receptor inhibited adenylate cyclase through a pertussis toxin-sensitive G-protein, and reduced forskolin-elevated cyclic adenosine monophosphate (cAMP) levels in response to agonists. The rank order of agonist potency was L(+)-2-amino-4-phosphonobutyric acid (L-AP4) > L-serine-O-phosphate > L-glutamate > quisqualate = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD). (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I) was a partial agonist at the hmGlu6 receptor, with a potency approaching that of L-serine-O-phosphate.