It has been suggested that presynaptic ionotropic glutamate receptors contribute to the regulation of synaptic transmission in the mammalian brain. Here, we recorded directly (34 °C) from nerve terminals in the hippocampus, the hippocampal mossy fiber boutons (MFBs). Local application of NMDA (100 microM) in the continued presence of D-Serine (20 microM) and 0.1 mM Mg2+ elicited a slowly rising and decaying inward current in MFBs. Presynaptic NMDA-evoked currents showed largest peak amplitude at a holding potential of -40 mV and a bell-shaped current-voltage relationship between 0 and -80 mV. NMDA-evoked currents were reduced by increasing extracellular Mg2+ to 1mM (at -40 mV) and largely blocked by bath application of the NMDA receptor antagonist D-APV (100 microM) suggesting that NMDA-evoked currents were mediated by presynaptic NMDA receptors. Presynaptic NMDA receptors were also activated by repetitive extracellular stimulation (20 stimuli, 100 Hz) in the stratum lucidum of the hippocampal CA3 region, most likely activating mossy fibers leading to synaptic glutamate release from neighboring MFBs. Presynaptic NMDA receptor-mediated currents showed a decay time constant of 1-2 s which is more than tenfold slower as observed in postsynaptic neurons suggesting differential functional properties between pre- and postsynaptic NMDA receptors in the hippocampal CA3 region.