Glutamate is the main excitatory neurotransmitter in the spinal cord and acts on several types of receptor, including N-methyl-d-aspartate (NMDA) receptors, which play an important role in synaptic plasticity and chronic pain. Three families of NMDA receptor subunit have been identified: NR1, NR2 (A–D) and NR3 (A and B). NMDA receptors are heteromeric channels that contain NR1 with at least one NR2 subunit. There is extensive evidence that NMDA receptors are present in spinal cord but little is known about their synaptic distribution. We have used an antigen-unmasking method involving pepsin treatment to reveal NR1, NR2A and NR2B subunits and have compared their distribution with that of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2 subunit, which is thought to be present at most glutamatergic synapses throughout the spinal cord. After pepsin treatment, punctate labelling was seen with antibodies against each of these subunits. Although NR1 puncta were present throughout the grey matter, NR2A was concentrated in laminae III–IV and NR2B in laminae I–II. The majority of puncta labelled with each NMDA receptor antibody were GluR2-immunoreactive, which suggests that they were present at synapses, and this was confirmed with electron microscopy for the NR1 and NR2A antibodies. However, many GluR2-immunoreactive puncta did not show NMDA receptor immunoreactivity. In laminae I–II, most NR2B puncta were also NR1-immunoreactive and a similar arrangement was found for NR2A/NR1 in laminae III–IV. These results suggest that many, but not all, glutamatergic synapses in the spinal cord possess NMDA receptors and that subunit composition varies in different regions.