The role of reactive oxygen species in causing DNA damage through interaction of chromium(III) and hydrogen peroxide was examined using plasmid relaxation assay and EPR spectroscopy. Marked DNA strand breakage was induced by CrCl 3 plus H 2O 2 in a phosphate buffer at pH 6–8.9; whereas, only slight DNA strand breakage was observed during similar treatment at pH less than 4. DNA breakage also increased as the reaction temperature and Cr(III)/H 2O 2 concentrations increased. Control experiments with Cr(III) or H 2O 2 alone did not cause DNA breakage. Sodium azide, D-mannitol, Tris-HCl, or catalase completely inhibited Cr(III)/H 2O 2-induced DNA breakage, but Superoxide dismutase did not. The D 2O enhancing effect on DNA breaks was not observed. Cr(III) pre-incubated with a 30-fold molar excess of EDTA did not cause any significant DNA breakage in the presence of H 2O 2. In a phosphate buffer containing Cr(III) and H 2O 2, singlet oxygen and hydroxyl radicals were detected using EPR spectrometry with the spin traps 2,2,6,6-tetramethyl-4-piperidone and 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), respectively. DMPO/ .OH adducts and DNA breakage induced by Cr(III)/H 2O 2 were markedly higher than those induced by Cr(VI)/H 2O 2. Furthermore, ascorbate decreased Cr(III)/H 2O 2-induced DNA breakage. EPR studies revealed that ascorbate (mole ratio to Cr( III) = 0.5:1) attenuated the DMPO/OH signal generated by Cr(III)/H 2O 2/DMPO, but a Cr(V) signal and ascorbate radicals were detected. NADPH, GSH, and GSSG also decreased DMPO/ .OH generated by Cr(III)/H 2O 2/ DMPO; however, they were less efficient than ascorbate and no Cr(V) signals were detected. This study shows that Cr(III)/H 2O 2 generates oxidative damage to DNA through a Fenton-like reaction: Cr(III) + H 2O 2 → Cr(IV) + .OH + OH − .