N-Methyl-D-aspartate receptors are fundamental for neuronal plasticity and development in the CNS. Most studies have examined postsynaptic roles of this receptor, but there are also indications for a presynaptic location and function. Here, we provide electrophysiological evidence for the existence of presynaptic N-methyl-D-aspartate receptors which can tonically facilitate glutamate release in the CNS. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoate reduced the frequency, but not amplitude, of glutamate-mediated spontaneous excitatory postsynaptic currents in layer II neurons of the rat entorhinal cortex. This effect was also observed in the presence of tetrodotoxin and when postsynaptic N-methyl-D-aspartate receptors were blocked by dialysis with dizocilpine maleate. When extracellular calcium was replaced with strontium, 2-amino-5-phosphonopentanoate reduced the "tail" of spontaneous excitatory postsynaptic currents that followed an evoked excitatory postsynaptic current. Finally, there was a tendency for paired-pulse facilitation of excitatory postsynaptic currents evoked at short (50 ms) intervals with postsynaptic N-methyl-D-aspartate receptors blocked) to be reduced by 2-amino-5-phosphonopentanoate, although this did not reach significance. These data strongly support the presence of presynaptic N-methyl-D-aspartate autoreceptors which may facilitate glutamate release in layer II of the entorhinal cortex.