Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level. However, the mechanism of cytotoxicity of this compound still remains controversial. This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione. Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells. GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains. These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells.