In vivo localized high-resolution 1H MR spectroscopy was performed in multiple brain regions without the use of anesthetic or paralytic agents in awake head-restrained rats that were previously trained in a simulated MRI environment using a 7T MR system. Spectra were obtained using a short echo time single-voxel point-resolved spectroscopy technique with voxel size ranging from 27 to 32.4 mm3 in the regions of anterior cingulate cortex, somatosensory cortex, hippocampus, and thalamus. Quantifiable spectra, without the need for any additional postprocessing to correct for possible motion, were reliably detected including the metabolites of interest such as γ-aminobutyric acid, glutamine, glutamate, myo-inositol, N-acetylaspartate, taurine, glycerophosphorylcholine/phosphorylcholine, creatine/phosphocreatine, and N-acetylaspartate/N-acetylaspartylglutamate. The spectral quality was comparable to spectra from anesthetized animals with sufficient spectral dispersion to separate metabolites such as glutamine and glutamate. Results from this study suggest that reliable information on major metabolites can be obtained without the confounding effects of anesthesia or paralytic agents in rodents. Magn Reson Med 69:937–943, 2013. © 2012 Wiley Periodicals, Inc.