The metabotropic glutamate receptors (mGluRs) have distinct distribution patterns in the CNS but subtypes within group I or group III mGluRs share similar ultrastructural localization relative to neurotransmitter release sites: group I mGluRs are concentrated in an annulus surrounding the edge of the postsynaptic density, whereas group III mGluRs are concentrated in the presynaptic active zone. One of the group II subtypes, mGluR2, is expressed in both pre- and postsynaptic elements, having no close association with synapses. In order to determine if such a distribution is common to another group II subtype, mGluR3, an antibody was raised against a carboxy-terminus of mGluR3 and used for light and electron microscopic immunohistochemistry in the mouse CNS. The antibody reacted strongly with mGluR3, but it also reacted, though only weakly, with mGluR2. Therefore, to examine mGluR3-selective distribution, we used mGluR2-deficient mice as well as wild-type mice. Strong immunoreactivity for mGluR3 was found in the cerebral cortex, striatum, dentate gyrus of the hippocampus, olfactory tubercle, lateral septal nucleus, lateral and basolateral amygdaloid nuclei, and nucleus of the lateral olfactory tract. Pre-embedding immunoperoxidase and immunogold methods revealed mGluR3 labeling in both presynaptic and postsynaptic elements, and also in glial profiles. Double labeling revealed that the vast majority of mGluR3 in presynaptic elements is not closely associated with glutamate and GABA release sites in the striatum and thalamus, respectively. However, in the spines of the dentate granule cells, the highest receptor density was found in perisynaptic sites (20% of immunogold particles within 60 nm from the edge of postsynaptic membrane specialization) followed by a decreasing receptor density away from the synapses (to approximately 5% of particles per 60 nm). Furthermore, 19% of immunogold particles were located in asymmetrical postsynaptic specialization, indicating an association of mGluR3 to glutamatergic synapses. The present results indicate that the localization of mGluR3 is rather similar to that of group I mGluRs in the postsynaptic elements, suggesting a unique functional role of mGluR3 in glutamatergic neurotransmission in the CNS.