Cu2+ ions and reduced glutathione (GSH) swiftly interact to form the physiologically occurring Cu(I)-[GSH]2 complex. Prompted by the recently reported ability of this complex to generate superoxide radicals from molecular oxygen, the present study addressed how the concentration of Cu 2+ and GSH, the pH, and the temperature affect the formation of the Cu(I)-[GSH]2 complex and its capacity to generate superoxide radicals and hydrogen peroxide. Increasing concentrations of Cu2+ and GSH, added at a fixed molar ratio of 1:3, led to a proportionally greater production of superoxide anions, hydrogen peroxide, and oxidized glutathione (GSSG). GSSG formation was found to closely reflect the formation of Cu(I)-[GSH]2. Biologically relevant changes in pH (e.g., from 6.8 to 7.7) and temperature (from 22 to 37 °C) did not affect the formation of the Cu(I)-[GSH] 2, as assessed by GSSG production. However, production of superoxide radicals increased as the pH values were incremented. An opposite effect was observed regarding hydrogen peroxide production. The ability of a freshly prepared Cu(I)-[GSH]2 complex (assayed within a minute from its formation) to generate superoxide radicals was incremented by as the temperature was increased. Such ability, however, correlated inversely with the temperature when, before assaying for superoxide, the earlier referred preparation was incubated during 30 min in the presence of oxygen. Under the latter condition, hydrogen peroxide linearly accumulated in time, suggesting that an increased autodismutation underlies the apparent time-dependent "aging" of the capacity of the complex to generate superoxide.