The glutathione (GSH)-mediated superoxide (O₂^•−) generation in an aqueous solution and relation of hydrogen peroxide (H₂O₂) and effect of catalase were investigated. GSH-induced O₂^•− generation in hyperthermal temperatures was measured by the nitroblue tetrazolium (NBT) mehod. Heating an aqueous solution containing GSH caused superoxide from dissolved O₂. H₂O₂ was generated simultaneously in this reaction mixture probably from the hydroperoxy radical (HO₂^•), which is equilibrated with O₂^•− in an aqueous condition, and then H₂O₂ consumed O₂^•−. Coexisting catalase in the reaction mixture, as a result, could increase O₂^•− generation. The catalase-exaggerated extracellular O₂^•− generation could give a harmful effect to living cells. This GSH-induced oxidative stress can be a part of mechanisms of hyperthermia therapy.