We tested the hypothesis that endogenous N-acetylaspartylglutamate (NAAG) presynaptically inhibits glutamate release at mossy fiber-CA3 synapses. For this purpose we made use of 2-(3-Mercaptopropyl)pentanedioic acid (2-MPPA), an inhibitor of glutamate carboxypeptidase II [GCP II; also known as N-acetylated alpha-linked acidic dipeptidase (NAALADase)], the enzyme that hydrolyzes NAAG into N-acetylaspartate and glutamate. Application of 2-MPPA (1 to 20 µM) had no effect on intrinsic membrane properties of CA3 pyramidal neurons recorded in vitro in whole cell current-clamp or voltage-clamp mode. Bath application of 10 µM 2-MPPA suppressed evoked EPSC amplitudes. Attenuation of EPSC amplitudes was accompanied by a significant increase in paired-pulse facilitation (50 msec inter-pulse intervals) suggesting that a presynaptic mechanism is involved. The group II metabotropic glutamate receptor (mGluR) antagonist LY341495 prevented the 2-MPPA-dependent suppression of EPSC amplitudes. 2-MPPA reduced the frequencies of TTX-insensitive miniature EPSCs (mEPSC), without affecting their amplitudes, further supporting a presynaptic action for GCP II inhibition. 2-MPPA-induced reduction of mEPSC frequencies was prevented by LY341495, reinforcing the role of presynaptic group II mGluR. Since GCP II inhibition is thought to increase NAAG levels, these results suggest that NAAG suppresses synaptic transmission at mossy fiber-CA3 synapses through presynaptic activation of group II mGluRs.