Isolation of the three new cytotoxic furonaphthaquinones 1, 2, and 3 (or 4) from T. cassinoides is described. The compounds were characterized by 1H nmr, uv, ir, and ms. The synthesis of quinone 1 is also reported. As part of a systematic survey of botanical sources for anticancer activity, an alcoholic extract of the stem bark of Tabebuia cassinoides (Lam.) DC. (Bignoniaceae) was examined. This extract showed slight but reproducible activity in the P-388 in vivo bioassay (T/C 127 and 125 at 200 mg/kg in two separate tests), and a full-scale investigation of it was thus initiated in an attempt to isolate the antileukemic constituent. Partitioning of the ethanol extract (54g) between chloroform and water gave a chloroform fraction (6.8 g) showing T/C 132 at 50 mg/kg in the P-388 assay. Partitioning of the chloroform fraction between 90% aqueous methanol and hexane yielded an inactive hexane fraction and a methanol-soluble fraction (3.6 g) with T/C 122 at 50 mg/kg and an ED60 of 27 pg/d in the KB cell culture assay. Further purification of the methanol fraction by chromatography on silica gel resulted in a loss of in vivo activity, and subsequent re-extractions of the original stem bark sample yielded extracts that were uniformly inactive in the P-388 in vivo assay. The first fraction eluted from the column showed significant cytotoxicity in the KB cell culture assay (ED50 1.9 pg/ml), however, and purification of this fraction by chromatography and recrystallization yielded the three new cytotoxic naphthaquinones 1-3 (or 4). Compound 1, C14H8O4, was an orange crystalline substance with an ED50 of 1.0 pg/ml in the KB cell culture assay. Its 1H nmr spectrum in CDCl3 was rather simple with two two-proton multiplets centered at δ 8.2 and 7.8 ppm, a one-proton singlet at 7.60 ppm, and a three-proton singlet at 2.70 ppm. Its ir spectrum showed absorptions at 1690 and 1670 cm-l and its uv spectrum was characteristic of napthaquinones. The occurrence of naphthaquinones in various members of the genus Tabebuia is well known, and lapachol (9) is one of the major constituents of several Tabebuia species (2). The biosynthesis of quinone 1 thus may occur via cyclization of lapachol to dehydr-iso-α-lapachone (13) (9), followed by oxidative cleavage of the exocyclic methylene group and dehydrogenation of the dihydrofuran ring. Quinones 2 and 3 (or 4) could then be derived by simple reductive and oxidative transformation of quinone 1. Although the three quinones are all relatively simple compounds, they have not previously been reported in the literature.