Acetate, a glial‐specific substrate, is an attractive alternative to glucose for the study of neuronal‐glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2‐¹³C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR_ace) increased progressively and reached close to saturation for plasma acetate concentration > 2–3 mM, whereas brain acetate concentration continued to increase. The Michaelis–Menten constant for brain acetate utilization ( = 0.01 ± 0.14 mM) was much smaller than for acetate transport through the blood–brain barrier (BBB) ( = 4.18 ± 0.83 mM). The maximum transport capacity of acetate through the BBB ( = 0.96 ± 0.18 μmol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization ( = 0.50 ± 0.08 μmol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2–3 mM. At such high plasma acetate concentration, the rate‐limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.