Reactive intermediates generated by phagocytes damage DNA and may contribute to the link between chronic inflammation and cancer. Myeloperoxidase, a heme protein secreted by activated phagocytes, is a potential catalyst for such reactions. Recent studies demonstrate that this enzyme uses hydrogen peroxide (H₂O₂) and nitrite (NO₂^-) to generate reactive nitrogen species which convert tyrosine to 3-nitrotyrosine. We now report that activated human neutrophils use myeloperoxidase, H₂O₂, and NO₂^- to nitrate 2‘-deoxyguanosine, one of the nucleosides of DNA. Through HPLC, UV/vis spectroscopy, and mass spectrometry, the two major products of this reaction were identified as 8-nitroguanine and 8-nitro-2‘-deoxyguanosine. Nitration required each component of the complete enzymatic system and was inhibited by catalase and heme poisons. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species that results from the one-electron oxidation of NO₂^- by myeloperoxidase. Alternatively, 2‘-deoxyguanosine might be oxidized directly by the enzyme to yield a radical species which subsequently reacts with NO₂^- or NO₂^• to generate the observed products. Human neutrophils stimulated with phorbol ester also generated 8-nitroguanine and 8-nitro-2‘-deoxyguanosine. The reaction required NO₂^- and was inhibited by catalase and heme poisons, implicating myeloperoxidase in the cell-mediated pathway. These results indicate that human neutrophils use the myeloperoxidase−H₂O₂−NO₂^- system to generate reactive species that can nitrate the C-8 position of 2‘-deoxyguanosine. Our observations raise the possibility that reactive nitrogen species generated by myeloperoxidase and other peroxidases contribute to nucleobase oxidation and tissue injury at sites of inflammation.