Cicuta marulata L. (Umbelliferae), known as spotted water-hemlock, is a stock-poisoning plant of North Carolina with reported human poisoning cases and historical use in herbal remedy for scirrhous mammary cancer and scirrhous tumors. As part of a search for novel antitumor agents, the methanolic extract of its whole plant showed significant in vitro cytotoxicity in the 9 KB (human nasopharyngeal carcinoma) cell culture assay. Bioassay-directed fractionation led to the isolation of cicutoxin (1) as the cytotoxic [ED50 (KB)=2.0 μg/ml] and antileukemic [T/C=165% against P-388 lymphocytic leukemia in mice at 1 mg/kg, 3-day dosing] principle. Cicutoxin was previously isolated from Cicuta virosa as a poisonous principle, but its potent antileukemic activity is revealed for the first time. Structural characterization of 1 (molecular formula C17H24O2) was based on hrms, ir, 13C-nmr (DEPT), formation of diacetate (2, C21H26O4) and saturated diol (3, C17H36O2), and 1H nmr data, with configuration at C-14 undetermined. Study of structure-cytotoxicity relationships using derivatives (2-7) showed all had ED50 (KB)>4.0 μg/ml and were less cytotoxic than 1, concluding that the potent activity of 1 is due to conjugated double/triple bonds and two hydroxyl groups.