The reduced form of glutathione (GSH; gamma-glutamyl cysteinyl glycine) is supposedly the most powerful reducing battery in the central nervous system against oxidative stress. We evaluated the contribution of GSH and GSSG to MEGA-PRESS (a frequency-selective refocusing technique) signals assessed by magnetic resonance spectroscopy (MRS). GSH gave a single positive signal (2.95 ppm) by the MEGA-PRESS. In contrast, GSSG gave a multiplet of reversed signals (3.03, 3.23, and 3.34 ppm). A phantom solution mimicking the normal in vivo condition (GSH:GSSG=100:1) gave a single positive peak. Even when the ratio was changed to 10:1, corresponding to toxic oxidative stress, GSH was prominent and GSSG signals were minimal. Thus, GSSG signals could be negligible. In the phantom solution (creatine:GSH:aspartate:gamma-aminobutyric acid=7:3:1:1), the creatine signal overshadowed the other signals. Through the MEGA-PRESS, a single peak of GSH stood out over other signals. In vivo, the brains of healthy volunteers gave similar signals as the in vitro phantom solution, indicating that the signal originated from GSH. The estimated concentration of GSH in the human brain was 1.9+/-0.37 mM (mean+/-S.D., n=4). In conclusion, MEGA-PRESS allowed us to assess GSH levels in vivo non-invasively.