2,3-Dimethyl-1,4-naphthohydroquinone undergoes auto-oxidation to the corresponding quinone at pH 7.4, with stoichiometric consumption of oxygen and formation of hydrogen peroxide. In an unpurified buffer, the rate of oxidation was low, but it increased nearly 9-fold when trace metals were removed from the buffer by treatment with Chelex resin. A similar increase in rate was achieved by addition of DTPA or bathophenanthroline sulfonate to unpurified buffer, whereas EDTA and desferal were less effective. Addition of copper to purified buffer led to inhibition of oxidation, with a 50% decrease in rate being observed at a metal concentration of 7.1 nM, and it is likely that the low auto-oxidation rate recorded in unpurified buffer was due to copper contamination of the latter. The auto-oxidation of 2,3-dimethyl-1,4-naphthohydroquinone was exceptionally sensitive to inhibition by superoxide dismutase, with a concentration of only 4.5 ng/ml being sufficient for a 50% decrease in rate, and the inhibitory effect of copper may be due to the ability of this metal to catalyse the dismutation of superoxide. Previous studies have shown that the rates of auto-oxidation of 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone are influenced by copper contamination of buffer and the present study shows that this is also true for a di-substituted naphthohydroquinone. For accurate assessment of rates of naphthohydroquinone auto-oxidation, it is important that purified buffers or appropriate chelating agents, are employed.