gamma-Irradiation of several amino acids (Val, Leu, Ile, Lys, Pro, and Glu) in the presence of O2 generates hydroperoxides. We have previously isolated and characterized valine and leucine hydroperoxides, and hydroxides, and have detected these products in both isolated systems [e.g., bovine serum albumin (BSA) and human low-density lipoprotein (LDL)] and diseased human tissues (atherosclerotic plaques and lens cataractous proteins). This work was aimed at investigating oxidized lysine as a sensitive marker for protein oxidation, as such residues are present on protein surfaces, and are therefore likely to be particularly susceptible to oxidation by radicals in bulk solution. HO* attack on lysine in the presence of oxygen, followed by NaBH4 reduction, is shown to give rise to (2S)-3-hydroxylysine [(2S)-2,6-diamino-3-hydroxyhexanoic acid], (2S)-4-hydroxylysine [(2S)-2,6-diamino-4-hydroxyhexanoic acid], (2S, 5R)-5-hydroxylysine [(2S,5R)-2,6-diamino-5-hydroxyhexanoic acid], and (2S,5S)-5-hydroxylysine [(2S,5S)-2,6-diamino-5-hydroxyhexanoic acid]. 5-Hydroxylysines are natural products formed by lysyl oxidase and are therefore not good markers of radical-mediated oxidation. The other hydroxylysines are however useful markers, with HPLC analysis of 9-fluorenylmethyl chloroformate (FMOC) derivatives providing a sensitive and accurate method for quantitative measurement. Hydroxylysines have been detected in the hydrolysates of peptides (Gly-Lys-Gly and Lys-Val-Ile-Leu-Phe) and proteins (BSA and histone H1) exposed to HO./O2, and subsequently treated with NaBH4. Quantification of the hydroxylysines yields, and comparison with hydroxyvalines and hydroxyleucines, supports the hypothesis that surface residues give higher yields of oxidized products than the hydrophobic leucines and valines, at least with globular proteins such as BSA. Hydroxylysines, and particularly 3-hydroxylysine, may therefore be sensitive and useful markers of radical-mediated protein oxidation in biological systems.