This study was part of a broader effort to identify and characterize promising atrazine-degrading phytochemicals in Eastern gamagrass (Tripsacum dactyloides ; EG) roots for the purpose of mitigating atrazine transport from agroecosystems. The objective of this study was to isolate and identify atrazine-degrading compounds in EG root extracts. Eastern gamagrass roots were extracted with methanol, and extracts were subjected to a variety of separation techniques. Fractions from each level of separation were tested for atrazine-degrading activity by a simple assay. Compounds were identified using high-performance liquid chromatography-tandem mass spectrometry. Results from the experiments identified 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) as the compound responsible for atrazine degradation in the root extract fractions collected. 2-β-d-Glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc) was also identified in the root extract fractions, but it did not demonstrate activity against atrazine. Estimated root tissue concentrations were 210 mg kg(-1) (wet wt basis) for DIBOA-Glc and 71 mg kg(-1) for HBOA-Glc (dry wt basis, 710 ± 96 and 240 ± 74 mg kg(-1), respectively). This research was the first to describe the occurrence and concentrations of an atrazine-degrading benzoxazinone compound isolated from EG tissue.