Glutamate together with its N-methyl-d-aspartate (NMDA) receptors has an important role in the transmission of stimuli in the spinal cord. Whilst the expression of the various NMDA receptor subunits within the spinal cord has been investigated the subcellular location of the NMDA NR2B subunit has yet to be definitively established. Both mRNA and light microscopical studies have failed to unequivocally demonstrate the proposed pre-synaptic location of this subunit. This has been proposed from pharmacological data and is thought to underlie the apparent analgesic properties of selective NR2B antagonists. Using pre-embedding immunohistochemistry combined with electron microscopy our findings provide the first definitive morphological evidence for both a pre- and post-synaptic localisation of NR2B/containing NMDA receptors, and suggest expression by astrocytes, in the rat lumbar spinal cord.