A group II metabotropic glutamate receptor (mGluR) agonist was recently reported to be clinically efficacious against positive and negative symptoms of schizophrenia (reference 1). The endogenous neuropeptide NAAG has been reported to have agonist activity at mGluR2 and mGluR3 (reference 2) and is degraded by the enzyme naaladase. Hence, elevating the concentration of NAAG by inhibition of naaladase represents a potential strategy for the treatment of schizophrenia via mGluR2/3 activation. Presynaptic mGluR2/3 autoreceptors modulate glutamatergic neurotransmission in the hippocampus (reference 3). We investigated the effect of NAAG and the naaladase inhibitor 2PMPA on paired field EPSPs (fEPSPs) evoked by stimulation of the perforant path and recorded in the dentate gyrus mid-molecular layer in adult Sprague-Dawley rat hippocampal slices. Bath application of NAAG (100 μM; n=6) had no effect on the amplitude of the fEPSP or the paired-pulse ratio while the mGluR2/3 agonist LY379268 (1 μM; n=5) inhibited the evoked fEPSPs and increased the paired-pulse ratio (Student t test, p<0.01), consistent with a presynaptic inhibitory action mediated by mGluR2/3 activation. In order to assess NAAG activity at group II mGluR more directly, we performed whole-cell voltage-clamp recordings from HEK MSRII cells transiently transfected with a human G protein-activated inwardly rectifying K⁺ channel concatamer (GIRK_1/2) and either human mGluR2 or mGluR3. Glutamate-elicited inward currents were observed with EC₅₀ of 18 μM and 1 μM for cells transfected with mGluR2 (n=3-5) and mGluR3 (n=3-4), respectively, and these were abolished by the mGluR2/3 antagonist LY341495 (1 μM; n=4). NAAG was purified using a cation exchange column and purity was confirmed by HPLC coupled with fluorescence detection analysis. Application of purified NAAG (300 μM or 1 mM) onto GIRK_1/2-mGluR2 or GIRK_1/2-mGluR3 transfected cells failed to evoke responses (n=4-5), whilst subsequent glutamate application (100 μM) induced LY341495-sensitive currents. To conclude, we found that purified NAAG had no agonist activity at group II mGluRs using the human recombinant mGluR2/3-GIRK_1/2 assay, consistent with our observations in rat hippocampal slices where NAAG and the naaladase inhibitor 2PMPA had no effect on glutamatergic transmission at the mGluR2/3-sensitive medial perforant pathway. Taken together, these findings do not support the rationale for targeting naaladase to increase brain NAAG levels as a therapeutic strategy for schizophrenia.