Acetate is a well-known astrocyte-specific substrate that has been used extensively to probe astrocytic function in vitro and in vivo. Analysis of amino acid turnover curves from ¹³C-acetate has been limited mainly for estimation of first-order rate constants from exponential fitting or calculation of relative rates from steady-state ¹³C enrichments. In this study, we used ¹H-[¹³C]-Nuclear Magnetic Resonance spectroscopy with intravenous infusion of [2-¹³C]acetate-Na⁺in vivo to measure the cerebral kinetics of acetate transport and utilization in anesthetized rats. Kinetics were assessed using a two-compartment (neuron/astrocyte) analysis of the ¹³C turnover curves of glutamate-C4 and glutamine-C4 from [2-¹³C]acetate-Na⁺, brain acetate levels, and the dependence of steady-state glutamine-C4 enrichment on blood acetate levels. The steady-state enrichment of glutamine-C4 increased with blood acetate concentration until 90% of plateau for plasma acetate of 4 to 5 mmol/L. Analysis assuming reversible, symmetric Michaelis–Menten kinetics for transport yielded 27±2mmol/L and 1.3±0.3 μmol/g/min for K_t and T_max, respectively, and for utilization, 0.17±0.24 mmol/L and 0.14±0.02 μmol/g/min for K_{M_util} and V_{max_util}, respectively. The distribution space for acetate was only 0.32±0.12 mL/g, indicative of a large excluded volume. The astrocytic and neuronal tricarboxylic acid cycle fluxes were 0.37±0.03 μmol/g/min and 1.41±0.11 μmol/g/min, respectively; astrocytes thus comprised ∼21%±3% of total oxidative metabolism.