The potency of L-glutamate in enhancing [3H]MK-801 binding to N-methyl-D-aspartate (NMDA) receptor ion channels was evaluated by quantitative autoradiography. In the presence of saturating concentrations of spermine, glycine and 20 microM D-2-amino-5-phosphonopentanoate (D-AP5), low concentrations of L-glutamate selectively increased [3H]MK-801 binding in the medial striatum and dorsal lateral septum. In comparison to these regions, EC50 values for L-glutamate stimulation of [3H]MK-801 binding were significantly higher in the cerebral cortex and thalamus. Regions displaying greater sensitivity to L-glutamate correlate to those regions where NMDA receptors are preferentially labelled by L-[3H]glutamate; regions less sensitive to L-glutamate correspond to regions selectively labelled by the NMDA antagonist [3H]CPP ([3H]3-((+) -2-carboxypiperazin-4-yl)-propyl-1-phosphonate). Since L-glutamate stimulation of [3H]MK-801 binding appears to reflect channel activation, these results suggest that it is possible to pharmacologically (or pathologically) activate anatomically-distinct subpopulations of NMDA receptors.