The history of certain Amaryllidaceous plants used in the primitive treatment of cancer can be traced to the fourth century B.C. In the genus Zephyranthes, Z. parulla Killip appears in a history of Peru (by Padre Cob) dated 1653, for treating tumors, and Z. rosea (Spreng.) Lindl. has found use in China for breast cancer (3). The leaves of Z. candida (Lindl.) Herb. have been employed in Africa as a treatment for diabetes mellitus (4); and the bulbs from a 1964 Hong Kong collection of this plant gave an extract that displayed a confirmed level of activity (cell line from a human epidermoid carcinoma of the nasopharynx, KB system) in the U. S. National Cancer Institute's (NCI) research programs (5). In this report we have summarized an investigation for antineoplastic constituents in the closely related Zephyranthes grandiflora Lindl., employing the NCI murine P-388 lymphocytic leukemia (PS system) for bioassay purposes. The bulbs of Z. grandiflora were extracted with CH2Cl2-MeOH-H2O, essentially as previously described for the Amaryllidaceous plant Pancratium littorale (2). Application of the solvent partitioning sequence 9:1-1:1 MeOH-H2O with hexane-CH2Cl2 to the CH2Cl2 phase of the original extract led to PS in vitro, but not in vivo, activity in the resulting CH2Cl2-MeOH-H2O fractions (Scheme 1). Because of our prior experience in locating the major PS in vivo active components of two other Amaryllidaceae species (2) in aqueous fractions, special emphasis was placed on that prospect. The aqueous MeOH phase of the bulb extract was concentrated, diluted with H2O, and extracted with n-BuOH (6). Separation of the n-BuOH fraction by partition chromatography on Sephadex LH-20 resulted in isolation of pancratistatin as the predominant PS in vivo and in vitro active (T/C 135%-150% at dose levels of 0.78-3.1 mg/kg, ED50 <0.01 μg/ml) constituent of Z. grandiflora. In our initial study (2) of pancratistatin (1), this promising isocarbostyril exhibited PS in vivo T/C 138%-165% at dose levels of 0.75-6.0 mg/kg and T/C 206% at 12.5 mg/kg. Based on the discovery of pancratistatin as a major antineoplastic biosynthetic product in species from two of three Amaryllidaceous genera we have so far examined, it would seem that this isocarbostyril and related substances (2) may account for some of the potentially valuable medicinal properties attributed to this plant family.