Leukocyte-induced DNA damage may partially account for the known association between chronic inflammation and malignancy. Since elucidation of the chemical nature of leukocyte-induced DNA damage may enhance our understanding of the mechanisms underlying leukocyte-induced DNA damage and the carcinogenesis associated with inflammation, the present study was undertaken to characterize the chemical modifications that occur in DNA exposed to stimulated human neutrophils. Calf thymus DNA was exposed to phorbol myristate acetate (PMA)-stimulated neutrophils in the presence or absence of exogenously added iron ions. DNA samples were subsequently hydrolyzed, derivatized and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring. A variety of base modifications including cytosine glycol, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine were identified. The yield of these various base products was increased by the addition of iron ions. Specifically, in the presence of physiologic quantities of iron ions, approximately 7 of every 1,000 DNA bases were modified. Addition of the superoxide anion scavenger, superoxide dismutase, the hydrogen peroxide scavenger, catalase, the hydroxyl scavenger, dimethylsulfoxide, or the iron chelator, deferoxamine, to DNA mixtures containing PMA, neutrophils, and iron ions, greatly decreased the yield of the damaged DNA base products. Our results indicate that stimulated human neutrophils can damage each of the four bases in DNA. It is likely that hydroxyl radical, generated via an iron catalyzed Haber-Weiss reaction, mediates neutrophil-induced DNA base damage, since: (a) the chemical structure of neutrophil-induced DNA base damage is consistent with a hydroxyl radical-mediated mechanism, (b) hydroxyl radical generated via ionizing radiation in aqueous solution produces DNA base modifications that are identical to neutrophil-induced DNA base modifications, (c) iron ions increase neutrophil-induced DNA base damage, and (d) iron chelators or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical decrease neutrophil-induced DNA base damage.