Specific binding sites for the noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, [3H]MK-801, were identified in synaptic membranes isolated from rat cerebellum. The density of these sites (0.61 pmol/mg protein), derived from linear Scatchard plots, was lower than those measured in a number of forebrain regions (0.81-2.96 pmol/mg protein). The Kd value for cerebellar [3H]MK-801 binding sites (37.7 nM) was markedly higher than those (1.53-1.82 nM) detected in rat forebrain regions. Experiments were carried out in the presence of 30 microM L-glutamic acid and 1 microM glycine, and after a 210 min incubation [3H]MK-801 binding was maximally stimulated. The pharmacology of these cerebellar [3H]MK-801 binding sites was markedly different from that of [3H]MK-801 sites in the rat cortex. These data have highlighted a novel population of NMDA receptors, which are functionally coupled to an ion channel but exhibit remarkably weak affinity for MK-801.