Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.