Proton magnetic resonance (MR) spectroscopy is a non-invasive technique used to determine relative concentrations of target metabolites in the brain. PURPOSE: The aim of this study was to determine the reliability of glutamate and GABA measures using a single-voxel proton MR spectroscopy protocol in healthy men and women. METHODS: Seven (4 female) healthy college age participants were tested at two time points separated by two weeks. The study was performed at 3T using a single voxel PRESS sequence for glutamate (TR/TE=1500/30ms) and an adapted MEGA-PRESS sequence for GABA (TR/TE=2000/68ms). Quantitation of glutamate and GABA was performed at two regions of interest, the primary motor cortex and the dorsolateral prefrontal cortex (DLPFC). Reliability across days was assessed with the intraclass correlation coefficient (ICC 2,1). RESULTS: There was no significant difference across time or region for glutamate (p=0.50 and p=0.06 respectively) or GABA (p=0.57 and p=0.91 respectively). There were no significant time-by-region interactions for glutamate (p=0.19) or GABA (p=0.26). Reliability across days was very strong for both glutamate (R=0.96) and GABA (R=0.88). CONCLUSIONS: These results support the use of MR spectroscopy measurements to reliably quantify concentrations of glutamate and GABA within the motor cortex and DLPFC.