Although several recent anatomical and physiological studies indicate that glutamate receptors are likely to play a role in the regulation of various hypothalamic functions, no attempt has yet been made to specifically characterize glutamate receptor densities, subtypes, or localization in the hypothalamus. To provide this basic information, we have characterized and mapped the binding of [3H]glutamate to N-methyl-D-aspartate (NMDA), non-NMDA, and metabotropic glutamate receptors throughout the diencephalon. Membrane binding assays revealed a [3H]glutamate binding density of 2.6 pmol/mg protein, approximately one third of the hippocampal density. Binding of subtype-specific agonists and antagonists was complex, but clearly indicated that each major glutamate subtype is present in all hypothalamic and preoptic regions in the following approximate relative densities: NMDA > metabotropic Glu receptor > kainate > or = alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid. Receptor autoradiography confirmed the widespread presence of all major glutamate receptor subtypes with roughly the following relative regional densities: ventromedial, dorsomedial > paraventricular, anterior hypothalamic, supraoptic > arcuate, suprachiasmatic, lateral hypothalamic > preoptic area >> pituitary neural lobe, white matter > pituitary anterior lobe (negligible). Subtype expression varied regionally, with rostral hypothalamic and preoptic regions having proportionally higher levels of non-NMDA vs. NMDA binding. High densities of glutamate receptors in ventromedial and medial hypothalamic regions suggest a prominent role in neuroendocrine and autonomic regulation.