Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging. (C) 2017 Elsevier Inc. All rights reserved.