The goal of this study was to investigate the effect of melatonin on basic cerebral metabolites in pinealectomized (Px) rat brains. Twenty-one rats were randomly divided into three groups with seven rats per group. The study groups included sham-operated rats, Px rats and Px rats treated with melatonin. Melatonin administration began at 60 days following pinealectomy and continued for 21 days. At the end of the study, in vivo single voxel magnetic resonance spectroscopy was performed on whole brains to determine choline (Cho), creatine and N-acetyl aspartate (NAA) concentrations. Px rats had significantly lower NAA levels (P < 0.05), and significantly higher Cho levels (P < 0.05) when compared with sham-operated rats. Administration of melatonin had normalized NAA and Cho levels in Px rats. We propose that pinealectomy causes significant changes in cerebral metabolites which are compatible with neural loss. Melatonin administration prevents the disruptive effects of pinealectomy on brain tissue.