AbstractBackground:  A nitroxide radical, 4‐hydroxyl‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl (TEMPOL), is directly reduced to hydroxylamine by ascorbic acid (AsA). Ascorbic acid is oxidized to dehydroascorbic acid (DHA) by ascorbic acid oxidase (AAOx), and DHA is reduced to AsA by glutathione (GSH). In the present study, in vivo and ex vivo reduction of TEMPOL in the rat liver under various conditions of AsA supply was investigated using an electron spin resonance (ESR) spectrometer equipped with a surface coil‐type resonator.Methods:  To investigate in vivo hepatic reduction of TEMPOL, an ESR study of the liver of living rats which orally received AsA or intravenously received GSH or AAOx was made. To investigate direct interactions between TEMPOL and GSH or AAOx, an in vitro ESR study was conducted. To investigate TEMPOL reduction in the hepatic homogenate, an ex vivo ESR study was performed.Results:  Ascorbic acid and GSH administration increased the in vivo hepatic reducing ability of TEMPOL. In contrast, AAOx administration decreased the reducing ability. In vitro TEMPOL was not reduced by GSH and hydroxylamine was not oxidized by AAOx. Reducing ability in the hepatic homogenate of AAOx‐treated rats decreased, but that for GSH‐treated rats was unchanged.Conclusion:  Ascorbic acid administration directly increases hepatic reducing ability. Ascorbic acid, which increased in the plasma due to GSH administration, entered the liver and enhanced the hepatic reducing ability. Administration of AAOx impaired the hepatic reducing ability by oxidizing AsA in the plasma and/or the liver.