Incubation of myeloperoxidase (MPO) with H2O2 in the presence of the spin trap DBNBS (3,5-dibromo-4-nitrosobenzenesulfonic acid) results in the EPR-detectable formation of a partially immobilized protein radical. The radical was only formed in the presence of both MPO and H2O2, indicating that catalytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, Like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441-35448], reacts with H2O2 to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent binding of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme.