After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-associated protein termed excitatory amino acid transporter 3 (EAAT3). Following entry into the nerve terminal, glutamate is pumped into synaptic vesicles by a vesicular transport system. Three different vesicular glutamate transporter proteins (VGLUT1-3) representing unique markers for glutamatergic neurons were recently characterized. The presence of EAAT3, glutaminase and VGLUT1-3 was examined in mouse, rat and rabbit species at mRNA and protein levels in hypothalamic neurons which are involved in the regulation of body weight using in situ hybridization and immunohistochemistry. EAAT3 and glutaminase mRNAs were demonstrated in all parts of the arcuate nucleus in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamic area. VGLUT1 mRNA was present in the magnocellular lateral hypothalamic nucleus. VGLUT2 mRNA was demonstrated in a subpopulation of neurons in the arcuate nucleus and in the ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamic area. Few VGLUT3 mRNA expressing neurons were scattered throughout the medial and lateral hypothalamus. EAAT3-like immunoreactivity (-li) was demonstrated in glutamate, neuropeptide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanin-concentrating hormone and orexin-immunoreactive (-ir) neurons. VGLUT2-li could only be demonstrated in POMC- and CART-ir neurons of the ventrolateral arcuate nucleus. The results show that key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NYP/AGRP neurons situated in the ventromedial part of the arcuate nucleus are mainly GABAergic, it is shown that several anorexigenic POMC/CART neurons of the ventromedial arcuate nucleus are most likely glutamatergic.