This paper examines processes in plants for the formation of fate products of TNT beyond its aminated reduction products, 2-amino-4,6-dinitrotoluene and 4-amino-2,6- dinitrotoluene. TNT metabolites were isolated and characterized in combination with temporal analyses of product profiles and 14C distribution, in microbe-free, axenic root cultures of Catharanthus roseus. Four unique TNTderived compounds were isolated. Using evidence from 1H NMR, mass spectroscopy, HPLC, acid hydrolysis, and enzymatic hydrolysis with â-glucuronidase and â-glucosidase, they were established as conjugates formed by reactions of the amine groups of 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. From the mass spectral evidence, at least a six-carbon unit from the plant intracellular millieu was involved in conjugate formation. Mass balance analysis indicated that, by 75 h after TNT amendment of the initial TNT radiolabel, extractable conjugates comprised 22%, bound residues comprised another 29%, 2-amino-4,6- dinitrotoluene was 4%, and the rest remained unidentified. Isolates from TNT-amended roots versus monoaminodinitrotoluene- amended roots were not identical, suggesting numerous possible outcomes for the plant-based conjugation of 2-amino-4,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene. This study is the first direct evidence for the involvement of the primary reduction products of TNTs2-amino-4,6- dinitrotoluene and 4-amino-2,6-dinitrotoluenesin conjugation processes in plant detoxification of TNT.