G protein-coupled receptors have a common structural motif of seven transmembrane alpha-helices and are classified into different families showing no sequence similarity. Extensive studies have been conducted on the structure-function relationship in family 1 receptors, but those in other families have not been well studied. In this study, to investigate the molecular basis leading to the G protein activation by metabotropic glutamate receptor (mGluR), the member of family 3, we searched for the amino acid residues responsible for the G protein activation in the second cytoplasmic loop, which was thought to be the main G protein binding region. Analyses of the systematical mutations of Gi/Go-coupled mGluR8 revealed the presence of a constitutively active mutation in the C-terminal region of the second loop. The corresponding mutation in the second loop of Gq-coupled mGluR1 also exhibited high agonist-independent activity. These results indicate that there is a common constitutive active mutation site regardless of mGluR subtypes, suggesting that the structural change of the junction between the second cytoplasmic loop and helix IV is strongly linked to the formation of the active state.