Three new cephalostatins possessing powerful cytostatic activity have been isolated from the Indian Ocean marine worm Cephalodiscus gilchristi; structures were assigned on the basis of detailed high-field n.m.r. and mass spectral analyses. Further bioassay (PS)-guided separation of C. gilchristi led to the isolation of three new steroidal alkaloids, designated cephalostatins 2, 3, and 4, similar in cell growth inhibition (PS E.D.50 10⁻⁷-10⁻⁹ yg ml⁻¹) to cephalostatin 1. A methylene chloride-methanol extract from 166 kg of wet C. gilchristi was separated by extensive solvent partition and gradient column chromatographic techniques. With the X-ray crystal structure of cephalostatin 1 as a reference, structures of cephalostatins 2-4 were unequivocally assigned via detailed analysis of high-field (400MHz) ¹H and ¹³C n.m.r. spectra, mass spectra, and elemental analyses. The thirteen fused or connected rings of the cephalostatins may be the largest such systems known in marine animals. Isolation and identification of cephalostatins 2-4 will allow initial assessment of structure-activity relationships in this series of vigorous cell growth inhibitors, with antineoplastic and other biological evaluations in progress.