The pivotal role of NMDA glutamate receptors in neuronal development and plasticity is dependent on their key properties of voltage-dependent Mg2+ block and Ca2+ permeability. In turn, these properties are primarily determined by the NR1, NR2 and NR3 subunit composition of the NMDA receptor. NR3 subunits confer radically different properties to NMDA receptors, reducing glutamate binding, voltage-dependent Mg2+ block and Ca2+ permeability. We have investigated the presence of NR3 subunits in the substantia nigra pars compacta where NMDA receptors influence the firing properties and plasticity of the predominantly dopaminergic neurone population. Rats aged postnatal day (P) 7 and P21 were perfusion-fixed with 2% formaldehyde (under halothane anaesthesia) for preparation of midbrain sections (50 μm, cryostat), containing the substantia nigra. Double immunolabelling using antibodies to NR3A or NR3B subunits and tyrosine hydroxylase (to identify dopaminergic neurones) was visualised on a confocal microscope using Alexa-conjugated secondary antibodies. Immunolabelling of both NR3A and NR3B subunits was observed in the substantia nigra. In P7 rats, 59% of 86 randomly sampled cells (from n=3 rats) expressed both tyrosine hydroxylase and NR3A; the remaining 41% expressed NR3A but not tyrosine hydroxylase. In P21 rats, 91% of 65 cells sampled expressed tyrosine hydroxylase, and all cells expressed NR3A. NR3B immunolabelling was also seen in substantia nigra cells expressing tyrosine hydroxylase in rats aged P7 (96% of 23 sampled cells; n=3 rats) and P21 (69% of 29 sampled cells; n=3 rats); the remaining cells expressed only NR3B. Therefore in substantia nigra dopaminergic neurones, NR3 subunits could form functional NR1/NR3 or NR1/NR2/NR3 NMDA receptors, which have distinct properties to NMDA receptors composed of NR1/NR2 subunits.