In previous studies, we demonstrated that the neuropeptide, N-acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1-6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD) and L-2-amino-4-phosphonobutyrate (L-AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans-ACPD, activated each of the transfected receptors, whereas L-AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans-ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.