NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2A/B-NR3A assemblies in adult human CNS. While NR1 and NR2A could only be efficiently solubilized by deoxycholate, NR3A was extracted by all detergents, suggesting that a large fraction is weakly anchored to cell membranes and other proteins. Using size exclusion chromatography we found that just as for NR1, a large fraction of NR3A exists as monomers and dimers, suggesting that these two glycine binding subunits behave similarly with regard to receptor assembly and trafficking.