The rate of (13)C-label incorporation into both aspartyl (NAA C3) and acetyl (NAA C6) groups of N-acetyl aspartate (NAA) was simultaneously measured in the rat brain in vivo for up to 19 h of [1-(13)C]glucose infusion (n = 8). Label incorporation was detected in NAA C6 approximately 1.5 h earlier than in NAA C3 because of the delayed labeling of the precursor of NAA C3, aspartate, compared to that of NAA C6, glucose. The time courses of NAA were fitted using a mathematical model assuming synthesis of NAA in one kinetic compartment with the respective precursor pools of aspartate and acetyl coenzyme A (acetyl-CoA). The turnover rates of NAA C6 and C3 were 0.7 +/- 0.1 and 0.6 +/- 0.1 micromol/(g h) with the time constants 14 +/- 2 and 13 +/- 2 h, respectively, with an estimated pool size of 8 micromol/g. The results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. Several hours after starting the glucose infusion, label incorporation into glutathione (GSH) was also detected. The turnover rate of GSH was 0.06 +/- 0.02 micromol/(g h) with a time constant of 13 +/- 2 h. The estimated pool size of GSH was 0.8 micromol/g, comparable to the cortical glutathione concentration. We conclude that NAA and GSH are completely turned over and that the metabolism is extremely slow (< 0.05% of the glucose metabolic rate).