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 C-14-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.