N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.