Past in vitro studies have used immunofluorescence to show increased clustering of the NR1 subunits of NMDA receptors (NMDAR) following NMDAR blockade, indicating that NMDARs self-regulate trafficking to and from spines. However, since a substantial portion of spinous NMDAR subunits can reside at sites removed from plasma membranes, whether or not these immunofluorescent clusters are synaptic remains to be shown. Also, the NR2A/B subunits undergo activity-dependent switching at synapses, indicating that their subcellular distribution may be regulated differently from the NR1 subunits. We examined the issue of NMDAR autoregulation by determining whether in vivo NMDAR blockade enhances trafficking of the NR2A subunits toward spines and more specifically to postsynaptic densities (PSDs) of already mature synapses. Seven adult rats received unilateral intra-cortical infusion of the NMDAR antagonist, D-AP5 for 1/2-2 h and the inactive enantiomer or the solvent, alone, in the contralateral cortex. Using an electron microscope, approximately 5600 cortical spines originating from the two hemispheres of the seven adult animals were analyzed for the location of NR2A subunits. In six out of the seven cases analyzed, the D-AP5-treated neuropil exhibited increased immunolabeling at PSDs and a concomitantly great increase at non-synaptic sites within spines. NR2A subunits also increased presynaptically within 1/2 h but not after 1 h. These findings indicate that NR2A subunits in intact, adult cortical neurons are prompted to become trafficked into spines and axon terminals by NMDAR inactivity, yielding an increase of a readily available reserve pool and greater localization at both sides of synapses.