Binding of the mGlu2/3 antagonist HYDIA in the closed conformation model of mGlu2 causes repulsive interactions with Y216 in lobe II of the binding pocket, preventing closure of the VFT.Modulation of metabotropic glutamate 2/3 receptors represents a promising target for the treatment of neuropsychiatric disorders such as schizophrenia and depression. The novel mGlu2/3 ligand HYDIA ((1S,2R,3R,5R,6S)-2-amino-3-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid) is a conformationally restricted and hydroxylated glutamate analogue. HYDIA is a potent and selective competitive antagonist of L-glutamate at the mGlu2/3 receptors in spite of being structurally very similar to the bicyclic LY354740, which is a potent and selective mGlu2/3 agonist. By comparing these two ligands, this study delineate the interaction mode of (3)H-HYDIA at the mGlu2 receptor, using both mutagenesis studies and computational modeling. Binding of HYDIA in the closed conformation model of mGlu2 results in repulsive interaction with the Y216 residue, preventing closure of the binding pocket and thus receptor activation. Consequently, HYDIA is proposed to bind in an open conformation model of mGlu2. Mutation of the structurally important Y216 residue in the binding site caused complete loss of affinity of both (3)H-LY354740 and (3)H-HYDIA. T168 in lobe I was shown to have an important role in HYDIA binding, and in the open conformation model this residue is interacting with the amino group of HYDIA. The Y144 residue in lobe I is shown to be engaged in both receptor interlobe binding and ligand interaction. Receptor mutations at this position (Y144G, Y144S and Y144A) showed dramatic impact on binding affinity and functional effect of HYDIA. The mGlu2 receptor mutants with increased structural flexibility at this position, which is crucial for pocket closure, were clearly preferred. These studies highlight the unique properties of the novel (3)H-HYDIA ligand and provide further support to our understanding of binding and signal transduction mechanisms of the mGlu2 receptor.