The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Gαq with the C-terminal five amino acids replaced with those from Gαi3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of l-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740> l - CCG- I= DCG- IV> l - glutamate≥(2R ,4 R)-APDC≥(1 S,3 R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was l-AP 4> l - SOP≥ ACPT- I= l - CCG- I≥ l - glutamate=(R , S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [ 35S]GTPγS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.