Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo 13C NMR spectroscopy coupled with the infusion labeled glial-specific substrate, such as acetate. In this study, we infused alpha-chloralose anesthetized rats with [2-13C]acetate and followed the dynamics of the fractional enrichment in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using 1H-[13C] MRS at 14.1T. Applying a two-compartment mathematical model to the measured time courses yielded a glial TCA cycle rate (Vg) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (VNT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (VPC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (Vxg and Vxn) were of the same order of magnitude as the respective TCA cycle fluxes. Additionally, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.