Oxidation of low density lipoprotein (LDL) may be of critical importance in triggering the pathological events of atherosclerosis. Myeloperoxidase, a heme protein secreted by phagocytes, is a potent catalyst for LDL oxidation in vitro, and active enzyme is present in human atherosclerotic lesions. We have explored the possibility that reactive intermediates generated by myeloperoxidase target LDL cholesterol for oxidation. LDL exposed to the myeloperoxidase. H 2O 2-Cl- system at acidic pH yielded a family of chlorinated sterols. The products were identified by mass spectrometry as a novel dichlorinated sterol, cholesterol α-chlorohydrin (6β- chlorocholestane-(3β,5α)-diol), cholesterol β-chlorohydrin (5α- chlorocholestane-(3β,6β)-diol), and a structurally related cholesterol chlorohydrin. Oxidation of LDL cholesterol by myeloperoxidase required H 2O 2 and C1 -, suggesting that hypochlorous acid (HOCI) was an intermediate in the reaction. However, HOCl failed to generate chlorinated sterols under chloride-free conditions. Since HOCI is in equilibrium with molecular chlorine (Cl 2) through a reaction which requires Cl - and H +, this raised the possibility that Cl 2 was the actual chlorinating intermediate. Consonant with this hypothesis, HOCl oxidized LDL cholesterol in the presence of Cl - and at acidic pH. Moreover, in the absence of Cl - and at neutral pH, Cl 2 generated the same family of chlorinated sterols as the myeloperoxidase- H 2O 2-Cl - system. Finally, direct addition of Cl 2 to the double bond of cholesterol accounts for dichlorinated sterol formation by myeloperoxidase. Collectively, these results indicate that Cl 2 derived from HOCl is the chlorinating intermediate in the oxidation of cholesterol by myeloperoxidase. Our observations suggest that Cl 2 generation in acidic compartments may constitute one pathway for oxidation of LDL cholesterol in the artery wall.