Among various metal ions of physiological interest, Cu(2+) is uniquely capable of catalyzing the oxidation of NADH by H2O2. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu(2+)-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H2O2 utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD(+). D2O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding(1)O2 as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H2O2.