Exceptionally potent biological properties exhibited by the sea hare Dolabella auricularia have been recorded for nearly two millennia. In 1972 we found Indian Ocean specimens of this captivating sea hare to yield extracts that proved very effective (over 100% increase in life span) against the US National Cancer Institute's (NCI) murine P388 lymphocytic leukemia (PS system). Subsequently, we isolated nine new (and powerful) cell growth inhibitory and/or antineoplastic peptides designated dolastatins 1-9 and two cytotoxic terpenes. Due to the dolastatins potency, the sea hare seems to require only vanishingly small quantities (ca. ~1 mg each from 100 kg), making isolation and structural elucidation of these peptides exceptionally challenging. Now we are pleased to report that our 15-year effort directed at discovering the most important Dolabella auricularia antineoplastic constituents has resulted in isolation and structural determination of a unique linear pentapeptide herein named dolastatin 10 (1). To our knowledge, dolastatin 10 is the most active (lowest dose) antineoplastic substance presently known and has shown, e.g., a 17-67% curative response at 3.25-26 pg/kg against the NCI human melanoma xenograph (nude mouse), 42-138% life extension at 1.44-11.1 pg/kg using the B16 melanoma, and 69-102% life extension at 1-4 pg/kg against the PS leukemia (ED50 4.6 × 10^-11 g/mL). A combined ethanol-2-propanol extract of D. auricularia (~1000 kg wet, collected in 1982) was concentrated to an active methylene chloride fraction by a series of solvent partition steps. Extensive column chromatographic separation (steric exclusion and partition on Sephadex, partition and adsorption on silica gel and HPLC) using gradient elution techniques guided by PS bioassay led to 28.7 mg of pure dolastatin 10 (1) as a colorless amorphous powder (from methylene chloride-methanol): C42H68N6O6S by HREIMS (M+ obsd. av. 784.4899, calcd for 784.4921); mp 107-112 °C; [α]D -68° (c 0.01, methanol); Rf 0.43 in 90:10:0.8:0.2 CH2Cl2-CH3OH-H2O-NH4OH; UV λmax (CH3OH) 216 (ε 20180) and 242 (ε 3609) nm. Vigorous hydrolysis of dolastatin 10 under acidic (6 N HCl; 110 °C; 24, 48, and 70 h) or basic (aqueous Ba(OH)2, 120 °C, 20 h) conditions followed by amino acid analyses of the products consistently gave evidence of valine and phenylalanine. The latter observation was not in complete accord with initial NMR spectral data and was a source of concern until a structure for the new masked Phe named dolaphenine was deduced. Meanwhile, dolastatin 10 was found refractory to simple acetylation and saponification reactions suggesting a cyclic peptide but eliminating a depsipeptide structure. Compelling evidence for a linear pentapeptide structure was obtained by partial hydrolysis (in addition to above acidic and basic conditions, hydrolysis with 1:1 concentrated HCl and propionic acid for 15 min at 160 °C was performed) followed by conversion of the products to N-trifluoroacetyl butyl esters corresponding to units Dov-Val, Dil, Dap, and Doe (see Figure 1) minus loss of the two methoxy groups (from Dil and Dap) and addition of water (to Dap). By means of detailed gas chromatographic separation followed by HREI mass spectrometry (Kratos MS-80, HREI- and CI-modes) the C-terminal unit Doe was found to lack a butyl ester and the N-terminal segment (Dov-Val) a trifluoroacetyl group. At this point extensive high resolution mass and 1H and 13C NMR (400 MHz using 1H-1H-COSY, 2D-J resolved, and 1H-13C-2D shift correlated methods) spectral studies (Table I) had already been analyzed and structures ascertained for the four hitherto unknown (in nature) amino acid constituents leaving only the correct sequence in question. The dolastatin 10 sequence was unequivocally assigned on the basis of SP-SIMS measurements in conjunction with the collision experiments.