Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N-Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA-specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte-type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.