Both N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) are localized almost exclusively to neurons, and have become important markers of neuronal viability in a number of cerebral pathological conditions. Using nuclear magnetic resonance spectroscopy combined with [1-13C]glucose administration (200 min infusion) we show that the synthesis of both NAA and NAAG can be observed. Label was incorporated into NAA from labeled acetate and from labeled aspartate, while NAAG was labeled from labeled glutamate. The low fractional enrichment of NAA (ca. 3%) relative to aspartate (20%) suggests a slow turnover rate, while NAAG (20.0%) and glutamate (25.2%) labeling were nearly equal, suggesting that NAAG labeling is near steady state. The rapid turnover of NAAG suggests an important role in glutamate delivery, while the slow rate of NAA turnover implies that its major role is as substrate for the formation of NAAG.