Degradation of TNT by bovine rumen fluid, a novel source of anaerobic microbes, was investigated. Whole rumen fluid contents were spiked with TNT and incubated for a 24 h time period. Supernatant samples taken at 0, 1, 2, 4, and 24 h were analyzed by reverse-phase HPLC with diode array detection. Within 1 h, TNT was not detectable and reduction products of TNT including 2-hydroxyl-amino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were present with smaller amounts of diamino-nitrotoluenes. Within 2 h, only the diamino and dihydroxyamino-nitrotoluene products remained. After 4 h, 2,4-diamino-6-nitrotoluene and 2,4-dihydroxyamino-6-nitrotoluene were the only known molecular species left. At 24 h known UV absorbing metabolites were no longer detected, suggesting further transformation such as complete reduction to triaminotoluene or destruction of the aromatic ring of TNT may have occurred. TNT was not transformed at 24 h in autoclaved and buffered controls. This study presents the first direct evidence of biodegradation of TNT by ruminal microbes. (C) 2004 Elsevier Inc. All rights reserved.