We have undertaken detailed studies of the mechanisms involved in the production of OH radicals during the copper-catalyzed oxidation of dithiothreitol (DTT). Most of these studies were conducted in real time, detecting based on its reaction with coumarin-3-carboxylic acid to produce the fluorescent derivative 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Studies of the kinetics of oxidizing DTT show that production of occurs in two stages: an initial lag period during which there is little production of 7-OHCCA, and a second reaction phase during which there is more rapid generation of. The duration of the initial reaction period depends on the concentrations of both DTT and. During this initial stage, oxygen consumption is high, although an increasing concentration of Cu²⁺ decreases the oxygen consumption. The rate of production of during the second phase of the reaction depends on the concentration of Cu²⁺, but little oxygen is consumed. A mechanism is proposed whereby a complex is formed and catalyzes oxidation of free DTT via formation of an oxygen-containing intermediate. The second phase of the reaction begins after complete oxidation of free DTT and involves production of and H₂ O₂ followed by generation of via reduction of H₂ O₂ by cuprous ion.