Objective-To determine relative concentrations of selected major brain tissue metabolites and their ratios and lobar variations by use of 3-T proton (hydrogen 1 [1H]) magnetic resonance spectroscopy (MRS) of the brain of healthy dogs. Animals-10 healthy Beagles. Procedures-3-T 1H MRS at echo times of 144 and 35 milliseconds was performed on 5 transverse slices and 1 sagittal slice of representative brain lobe regions. Intravoxel parenchyma was classified as white matter, gray matter, or mixed (gray and white) and analyzed for relative concentrations (in arbitrary units) of N-acetylaspartate (NAA), choline, and creatine (ie, height at position of peak on MRS graph) as well as their ratios (NAA-to-choline, NAA-to-creatine, and choline-to-creatine ratios). Peak heights for metabolites were compared between echo times. Peak heights for metabolites and their ratios were correlated and evaluated among matter types. Yield was calculated as interpretable voxels divided by available lobar voxels. Results-Reference ranges of the metabolite concentration ratios were determined at an echo time of 35 milliseconds (NAA-to-choline ratio, 1.055 to 2.224; NAA-to-creatine ratio, 1.103 to 2.161; choline-to-creatine ratio, 0.759 to 1.332) and 144 milliseconds (NAA-to-choline ratio, 0.687 to 1.788; NAA-to-creatine ratio, 0.984 to 2.044; choline-to-creatine ratio, 0.828 to 1.853). Metabolite concentration ratios were greater in white matter than in gray matter. Voxel yields ranged from 43% for the temporal lobe to 100% for the thalamus. Conclusions and Clinical Relevance-Metabolite concentrations and concentration ratios determined with 3-T 1H MRS were not identical to those in humans and were determined for clinical and research investigations of canine brain disease.