Glutamate neurotransmission involves numerous ionotropic and metabotropic glutamate receptor types in postsynaptic, presynaptic and glial locations. Distribution of the metabotropic glutamate receptors mGluR2 and mGluR3 was studied with an affinity-purified, characterized polyclonal antibody made from a C-terminus peptide. This antibody, mGluR2/3, recognized both mGluR2 and mGluR3, but did not cross-react with any other type of metabotropic glutamate receptor except for a very slight recognition of mGluR5. Light microscope distribution of the antibody binding sites in the nervous system matched the combined distributions of messenger RNA for mGluR2 and mGluR3. For example, dense staining seen in the accessory olfactory bulb and cerebellar Golgi cells matched high levels of mGluR2 messenger RNA in these structures, while moderately dense staining in the reticular nucleus of the thalamus and light to moderate staining in glia throughout the brain matched significant levels of mGluR3 messenger RNA in these structures. In the rostral olfactory structures, the densest stained neurons belonged to presumptive "necklace olfactory glomeruli." In the hippocampus, staining was densest in the neuropil of the stratum lucidum/pyramidale, stratum lacunosum/moleculare, hilus and middle third of the molecular layer of the dentate gyrus. Ultrastructural studies of the cerebral cortex, hippocampus and caudate-putamen revealed significant staining in postsynaptic and presynaptic structures and glial wrappings of presumptive excitatory synapses; frequently, this staining was concentrated in discrete patches at or near active zones. In the hippocampus, presynaptic staining appeared to be concentrated in terminals of two populations of presumptive glutamatergic axons: mossy fibers originating from granule cells and perforant path fibers originating from the entorhinal cortex. These data suggest that populations of mGluR2 and/or mGluR3 receptors are localized differentially in synapses, i.e. those in and near the postsynaptic and presynaptic membranes and in glial wrappings of synapses, in several regions of the brain. In addition, we provide immunocytochemical evidence of mGluR2 or mGluR3 receptors in presynaptic terminals of glutamatergic synapses. Thus, mGluR2 and mGluR3 are found in various combinations of presynaptic, postsynaptic and glial localizations that may reflect differential modulation of excitatory amino acid transmission.