Localized in vivo 1H magnetic resonance spectroscopy (MRS) was used to investigate metabolite levels in the brain of adult Zucker Diabetic Fatty (ZDF) rats, an animal model for type 2 diabetes mellitus. This study focussed on the hippocampus, assumed to be one of the main brain areas affected by this disease. Together with an almost 5-fold increase in blood glucose concentration measured by glucose oxidation, significant increases were found in the hippocampal concentrations of glucose (4.93 vs 1.66 mM p < 0.001), myo-inositol (6.52 vs 4.30 mM; p < 0.05), and total creatine (12.71 vs 10.50 mM; p < 0.05) in ZDF rats (n = 5) compared with littermates (n = 5). Although no obvious alterations were detected in the hippocampal levels of other metabolites, including NAA + NAAG and choline-containing compounds in the ZDF rats, the increase in Glc and Ins levels is in line with elevated brain tissue contents of these metabolites in patients with diabetes mellitus.