Copper toxicity has been presumed to involve catalytic hydroxyl radical (.OH) formation from hydrogen peroxide. Addition of Cu1+ to a solution containing ethanol or dimethylsulfoxide (Me2SO) and the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) results in formation of the alpha-hydroxyethyl radical or methyl radical adduct of 4-POBN, respectively. Adduct formation was prevented by inclusion of catalase, but not by superoxide dismutase. Inclusion of exogenous H2O2 in the reaction mixture increased the yield of ethanol- or Me2SO-derived radical adduct and also enhanced the formation of secondary radical adducts, including 4-POBN/.H and the methyl radical adduct of 2-methyl-2-nitrosopropane. The alpha-hydroxyethyl adduct of 4-POBN is rapidly decomposed in the presence of copper, but not iron salts, whereas the methyl radical adduct is relatively stable in the presence of inorganic copper. The total concentration of radical adduct detected from the reaction between Cu1+ and H2O2, determined by comparison of the integrated spectral intensity with that of the stable 2,2,6,6-tetramethyl-1-piperidinyloxy free radical, was only 1-5% of the maximum amount predicted assuming radical adduct formation from all of the added copper. A variety of copper chelators inhibit formation of carbon-centered radical adducts of 4-POBN, including penicillamine and triethylenetetramine, which are the primary drugs used to treat the copper metabolism disorder Wilson's disease. The results provide clear evidence for hydroxyl radical formation from Cu1+ and H2O2 (either added or formed during the autoxidation of reduced copper.