The hypothalamic arcuate nucleus contains a number of neurochemically different cell populations, among others neuropeptide Y (NPY)- and pro-opiomelanocortin (POMC)-derived peptide-expressing neurons; both are involved in the regulation of feeding and energy homeostasis, NPY neurons also in the release of hypophysiotropic hormones, sexual behaviour and thermogenesis. Recent observations indicate that there is a dense plexus of glutamatergic fibres in the arcuate nucleus. The aim of the present studies was to examine the relationship of these fibres to the NPY and POMC neurons in the arcuate nucleus. Double-label immunoelectron microscopy was used. Glutamatergic elements were identified by the presence of vesicular glutamate transporter 1 (VGluT1) or 2 (VGluT2) (selective markers of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive terminals was observed to make asymmetric type of synapses with NPY and with β-endorphin (a marker of POMC neurons)-immunostained nerve cells of the arcuate nucleus. About 15% of VGluT2 synapsing terminals established asymmetric synapses with NPY-positive cells and more than 40% of VGlut2-positive terminals formed synapse on β-endorphin-positive neurons. VGluT2-positive perikarya were also observed, part of them also contained β-endorphin. Nerve terminals containing both VGluT2 and β-endorphin were demonstrated in the cell group. Only very few VGluT1 fibres were detected. Our observations provide the first direct neuromorphological evidence for the existence of glutamatergic innervation of NPY and POMC neurons of the arcuate nucleus.