Incubation of cultured Chinese hamster V79 cells with menadione (2-methyl-1,4-naphthoquinone), a generator of superoxide anion radicals, caused a rapid increase in the level of glutathione disulfide (GSSG) and a decrease in the level of glutathione (GSH), which followed a 1.5- to 2-fold increase in the level of GSH during post-treatment incubation. Menadione also caused a concentration- and time-dependent increase in the activity of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme in the synthesis of GSH. These results suggested that the increase in level of GSH after treatment with menadione was due to the increase in the activity of gamma-GCS. Dicoumarol, an inhibitor of DT-diaphorase, did not influence the increase in the activity of gamma-GCS caused by menadione but it did enhance the cytotoxicity and the increase in the level GSSG caused by menadione. This result suggested that neither the DT-diaphorase-mediated metabolism of menadione nor the increase in level of GSSG caused by menadione was associated with the increase in the activity of gamma-GCS. Chelators of divalent iron and copper (I), and cycloheximide did not influence the increase in the activity of gamma-GCS caused by menadione. Thus, it appeared that reactive oxygen radicals, generated from hydrogen peroxide by an iron- or copper-catalyzed Fenton reaction, were not responsible for the increase in the activity of gamma-GCS and that the increase was not an inducible phenomenon.