Basic knowledge of the plant transformation pathways and toxicity of 2,4-dinitrotoluene (2,4-DNT) will assist in the design and assessment of a phytoremediation strategy. This study presents the toxicity and fate of 2,4-DNT and gene expression in response to 2,4-DNT exposure using the model plant Arabidopsis thaliana, an increasingly popular system for genetic and biochemical studies of phytotransformation of explosives. From the results of biomass and root growth assays for toxicity, 2,4-DNT was toxic to the plants at concentrations as low as 1 mg/L. In the uptake study, 95% of the initial 2,4-DNT was removed by 15-day-old seedlings from liquid media regardless of the initial 2,4-DNT concentrations while 30% accounted for the adsorption to the autoclaved plant materials. The mass balance was over 86% using [U-C-14]2,4-DNT, and the mineralization by the plants was less than 1% under sterile conditions during 14 days of exposure. The percentage of the bound radioactivity increased from 49% to 72% of the radioactivity in the plants, suggesting transformed products of 2,4-DNT may be incorporated into plant tissues such as lignin and cellulose. Monoaminonitrotoluene isomers and unknown metabolites with short retention times were detected as transformed products of 2,4-DNT by the plants. Most (68%) of the radioactivity taken up by the plants was in the root tissues in nonsterile hydroponic cultures. Glutathione and expression of related genes (GSH1 and GSH2) in plants exposed to 2,4-DNT were 1.7-fold increased compared to untreated plants. Genes of a glutathione S-transferase and a cytochrome P450, which were induced by 2,4,6-trinitrotoluene exposure in previous studies, were upregulated by 10- and 8-fold, respectively. The application of phytoremediation and the development of transgenic plants for 2,4-DNT may be based on TNT phytotransformation pathway characteristics because of the similar fate and gene expression in plants.