A phytotoxin that inhibited seedling root growth was isolated and purified from rhizomes of quackgrass [Agropyron repens (L.) Beauv.]. Aqueous solutions of the phytotoxin (0.1%, w/v) significantly inhibited the seedling root growth of corn (Zea mays L. ‘New Jersey 8’), oat (Avena sativa L. ‘Clintland 60’), cucumber (Cucumis sativus L. ‘Straight Eight’), and alfalfa (Medicago sativa L. ‘Vernal’). Growth inhibition was not due to extremes of pH or osmotic potential of the inhibitor solutions. The phytotoxin was isolated and purified from a methanol:water extract of dried and ground rhizomes by adsorption and elution from charcoal. A golden-brown, crystalline substance was obtained that was soluble in water, partly soluble in semipolar solvents, and insoluble in nonpolar solvents. Thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) suggested that a single compound was present. Elemental analysis showed that it contained 42.17% carbon, 5.06% hydrogen, 38.28% oxygen, 4.02% nitrogen, 2.03% sulfur, 0.03% chlorine, and 0.03% phosphorus. The compound was tentatively identified as a glycoside with a molecular weight of 460. Its ultraviolet (UV) absorption spectrum showed an intense maximum at 307 nm.