Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H2O2 and Cl- to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2'- deoxycytidine to the myeloperoxidase-H2O2-Cl- system, we obtained a single major product that was identified as 5-chloro-2'-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2'-deoxycytidine production by myeloperoxidase required H2O2 and Cl-, suggesting that HOCl is an intermediate in the reaction. However, reagent HOCl failed to generate 5- chloro-2'-deoxycytidine in the absence of Cl-. Moreover, chlorination of 2'- deoxycytidine was optimal under acidic conditions in the presence of Cl-. These results implicate molecular chlorine (Cl2), which is in equilibrium with HOCl through a reaction requiring Cl- and H+, in the generation of 5- chloro-2'-deoxycytidine. Activated human neutrophils were able to generate 5- chloro-2'-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H2O2-Cl- system generated similar levels of 5- chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell- associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl2 derived from HOCl generates 5-chloro-2'- deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2'- deoxycytidine. Phagocytic generation of Cl2 therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.