We have previously shown that presynaptic N-methyl-D-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit-selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca2+ entry through either NMDARs or voltage-gated Ca2+ channels with Co2+ prevented the effects of HQA, confirming that Ca2+ entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.