Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.