Magnocellular neuroendocrine cells of the supraoptic nucleus (SON) release the peptides oxytocin (OT) and vasopressin (VP) from their dendrites and terminals. In addition to peptide-containing large dense-core vesicles, axon terminals from these cells contain clear microvesicles that have been shown to contain glutamate. Using multilabelling confocal microscopy, we investigated the presence of vesicular glutamate transporters (VGLUTs) in astrocytes as well as VP and OT neurones of the SON. Simultaneous probing of the SON with antibodies against VGLUT isoforms 1-3, OT, VP and glial fibrillary acidic protein (GFAP) revealed the presence of VGLUT-2 in somata and dendrites of SON neurones. Immunoreactivity (-ir) for VGLUT-3 was also detected in both OT and VP neurones as well as in GFAP-ir astrocytes and other cells of the ventral glial lamina. Colocalisation of VGLUT-2 and VGLUT-3 in individual SON neurones was also examined and VGLUT-ir with both antibodies could be detected in both types of SON neurones. Although VGLUT-1-ir was strong lateral to the SON, only sparse labelling was apparent within the nucleus, and no colocalisation with either SON neurones or astrocytes was observed. The SON or the SON plus its surrounding perinuclear zone was probed using the reverse transcriptase-polymerase chain reaction and the presence of mRNA for all three VGLUT isoforms was detected. These results suggest that similar arrangements of transmitters exist in SON neuronal dendrites and their neurohypophysial terminals and that magnocellular neuroendocrine somata and dendrites may be capable of glutamatergic transmission. © 2006 The Authors. Journal compilation 2006 Blackwell Publishing Ltd.