Xanthium strumarium L. (Compositae, Heliantheae) is a spineless robust plant which occurs widely in America; but it is also naturalized in the waste places and tracksides of the Mediterranean countries. The plant is known in Greek as "agriovaxia"; the decoction of the resin has been used as a herbal remedy for the treatment of cancer. It is poisonous to livestock and causes also paralysis. The toxicity and the use of the plant suggest the probable presence of cytotoxic compounds. As a consequence, we have undertaken a search for the active compounds responsible for the in vitro and in vivo cytotoxicity of X. strumarium. The bioassay-directed fractionation of the active CH₂Cl₂ soluble residue led to the isolation and characterization of xanthatin (a seco-4,5-guaianolide). Xanthatin was tested against the two murine lymphocytic leukemia cell-lines P-388 and L-1210 and it showed even higher cytotoxicity (IC₅₀ = 0.018 µg/ml and 0.009 µg/ml, respectively) compared to 6-mercaptopurin as standard drug. It also exhibited activity against the resistant human origin-bronchial epidermoid carcinoma NSCLC-N6 (IC₅₀ = 3 µg/ml). In vivo test on murine ascite leukemia P-388 showed weak activity but low toxicity (LD₅₀ = 800 mg/kg). This is the first time that the in vitro and in vivo activity of xanthatin is described. The interesting cytotoxicity together with the low toxicity suggest a further exploration on the cytotoxic activity of this compound or its synthetic derivatives.