The metabotropic glutamate receptor mGluR1 is a G-protein-coupled glutamate receptor whose activation induces phosphotidylinositol hydrolysis and increases diacylglycerol and cytoplasmic calcium. By using affinity-purified antisera against a partial amino acid sequence of mGluR1 alpha, deduced from the nucleotide sequence of the cloned gene, the heterogeneous expression of this glutamate receptor was studied immunocytochemically with light and electron microscopy in the rat hypothalamus. Immunoreactivity was restricted to cell bodies and dendrites throughout many regions of the adult hypothalamus, including the preoptic area, anterior hypothalamus, suprachiasmatic nucleus, dorsomedial hypothalamus, and periventricular region. Strong immunolabeling was found in the lateral hypothalamus where immunoreactivity could be detected as early as embryonic day 18. Intense immunoreactivity was also found in the medial mammillary nuclei. In contrast to the strong labeling in many other regions, the neuroendocrine neurons of the arcuate, supraoptic, and paraventricular nuclei showed relatively little staining in adults. With light microscopy, immunoperoxidase labeling was found distributed in patches on the cytoplasmic side of the plasma membrane of immunoreactive neurons. When the same tissue was examined ultrastructurally, the patches were not restricted to synaptic specializations but were also found distributed on perikaryal and dendritic membranes sometimes associated with synapses and sometimes not. Some immunoreactive membranes showed no immunolabeling at the synaptic junction. When the tissue was strongly stained, labeling could be found in the cytoplasm of immunoreactive cells. No immunostaining was found on axons or presynaptic boutons. Together with other evidence showing a widespread expression of many different subtypes of both ionotropic and metabotropic receptors, these data support the hypothesis that glutamate may regulate hypothalamic cellular activity with a number of physiologically different mechanisms, and these mechanisms include second-messenger systems activated by G proteins.