Biodegradation of 2,4,6-trinitrotoluene (TNT) by the wood-rotting Basidiomycete Phanerochaete chrysosporium was studied in a fixed-film silicone membrane bioreactor and in agitated pelleted cultures. The initial intermediate products of TNT biodegradation were shown to be 2-amino-4,6-dinitrotoluene (2amDNT) and 4-amino-2,6-dinitrotoluene (4amDNT). These intermediates were also degraded by P. chrysosporium. However, their rates of degradation were slow and appeared to represent rate-limiting steps in TNT degradation. The fact that 2amDNT and 4amDNT were further degraded is of importance. In most other microbial systems these compounds are typically not further degraded or are dimerized to even more persistent azo and azoxydimers. Similar to previous studies performed in stationary cultures, it was shown that substantial amounts of [C-14]-TNT were degraded to [C-14]-carbon dioxide in agitated pelleted cultures. Lignin peroxidase activity (assayed by veratryl alcohol oxidation) virtually disappeared upon addition of TNT to ligninolytic cultures of P. chrysosporium. However, TNT, 2amDNT, and 4amDNT did not inhibit lignin peroxidase activity, nor were they substrates for this enzyme. Subsequent studies revealed that 4-hydroxylamino-2,6-dinitrotoluene, an intermediate in TNT reduction, was a potent lignin peroxidase inhibitor. Further studies revealed that this compound was also a substrate for lignin peroxidase H8.