Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.