Synaptic depression of evoked EPSCs was quantified with stimulation frequencies ranging from 0.2 to 100 Hz at the single CNS synapse formed by the calyx of Held in the rat brainstem. Half-maximal depression occurred at approximately 1 Hz, with 10 and 100 Hz stimulation frequencies reducing EPSC amplitudes to approximately 30% and approximately 10% of their initial magnitude, respectively. The time constant of recovery from depression elicited by 10 Hz afferent fiber stimulation was 4.2 sec. AMPA and NMDA receptor-mediated EPSCs depressed in parallel at 1-5 Hz stimulation frequencies, suggesting that depression was induced by presynaptic mechanism(s) that reduced glutamate release. To determine the contribution of autoreceptors to depression, we studied the inhibitory effects of the metabotropic glutamate receptor (mGluR) agonists (1S, 3S)-ACPD and L-AP4 and found them to be reversed in a dose-dependent manner by (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), a novel and potent competitive antagonist of mGluRs. At 300 microM, CPPG completely reversed the effects of L-AP4 and (1S, 3S)-ACPD, but reduced 5-10 Hz elicited depression by only approximately 6%. CPPG-sensitive mGluRs, presumably activated by glutamate spillover during physiological synaptic transmission, thus contribute on the order of only 10% to short-term synaptic depression. We therefore suggest that the main mechanism contributing to the robust depression elicited by 5-10 Hz afferent fiber stimulation of the calyx of Held synapse is synaptic vesicle pool depletion.