Production of O-2(-) by manganese peroxidase (MnP) in the absence of free radical intermediates was analyzed by ESR spin trapping experiments with 4-POBN and DMPO. MnPs from Pleurotus ostreatus and Bjerkandera adusta produced O-2(-) after addition of an excess of H2O2 independently of the presence of organic acid chelator for Mn(II) and Mn(III). This is the first report demonstrating direct production of O-2(-) by MnP.