A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2− and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.