Under ligninolytic conditions, the white-rot basidiomycete Coriolus versicolor metabolized chloronitrofen (2, 4, 6-trichloro-4'-nitrodiphenyl ether; CNP) and nitrofen (2, 4-dichloro-4'-nitrodiphenyl ether; NIP), which constitute the largest class of commercially produced diphenyl ether herbicides. The pathway of CNP degradation was elucidated by the identification of fungal metabolites upon addition of CNP and its metabolic intermediates. The metabolic pathway was initially branched to form four metabolites - 2, 4, 6-trichloro-3-hydroxy-4'-nitrodiphenyl ether, 2, 4-dichloro-6-hydroxy-4'-nitrodiphenyl ether, NIP, and 2, 4, 6-trichloro-4'-aminodiphenyl ether - indicating the involvement of hydroxylation, oxidative dechlorination, reductive dechlorination, and nitro-reduction. Of these reactions, hydroxylation was relatively major compared to the others. Extracellular ligninolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase did not catalyze the oxidation of either CNP or NIP. Piperonyl butoxide, an inhibitor of cytochrome P450, suppressed fungal oxidation of CNP and NIP to their hydroxylated products. The inhibition resulted in increasing the amount of reductively dechlorinated and nitro-reduced products. These observations strongly suggest that basidiomycetes may possess a mechanism for a strict substrate recognition system and a corresponding metabolic response system to effectively degrade environmentally persistent aromatic compounds.