Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM). was able to mineralize 14C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS*) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS* was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS* in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP.