Acetate has been proposed as an astrocyte-specific energy substrate for metabolic studies in the brain. The determination of the relative contribution of the intracellular and extracellular compartments to the acetate signal using diffusion-weighted magnetic resonance spectroscopy can provide an insight into the cellular environment and distribution volume of acetate in the brain. In the present study, localized (1) H nuclear magnetic resonance (NMR) spectroscopy employing a diffusion-weighted stimulated echo acquisition mode (STEAM) sequence at an ultra-high magnetic field (14.1 T) was used to investigate the diffusivity characteristics of acetate and N-acetylaspartate (NAA) in the rat brain in vivo during prolonged acetate infusion. The persistence of the acetate resonance in (1) H spectra acquired at very large diffusion weighting indicated restricted diffusion of acetate and was attributed to intracellular spaces. However, the significantly greater diffusion of acetate relative to NAA suggests that a substantial fraction of acetate is located in the extracellular space of the brain. Assuming an even distribution for acetate in intracellular and extracellular spaces, the diffusion properties of acetate yielded a smaller volume of distribution for acetate relative to water and glucose in the rat brain.