The complex marine natural product halichondrin B was compared with NSC 707389 (E7389), a structurally simplified, synthetic macrocyclic ketone analog, which has been selected for clinical trials in human patients. NSC 707389 was invariably more potent than halichondrin B in its interactions with tubulin. Both compounds inhibited tubulin assembly, inhibited nucleotide exchange on -tubulin, and were noncompetitive inhibitors of the binding of radiolabeled vinblastine and dolastatin 10 to tubulin. Neither compound seemed to induce an aberrant tubulin assembly reaction, as occurs with vinblastine (tight spirals) or dolastatin 10 (aggregated rings and spirals). We modeled the two compounds into a shared binding site on tubulin consistent with their biochemical properties. Of the two tubulin structures available, we selected for modeling the complex of a stathmin fragment with two tubulin heterodimers with two bound colchicinoid molecules and a single bound vinblastine between the two heterodimers (Nature (Lond) 435: 519 -522, 2005). Halichondrin B and NSC 707389 fit snugly between the two heterodimers adjacent to the exchangeable site nucleotide. Fitting the compounds into this site, which was also close to the vinblastine site, resulted in enough movement of amino acid residues at the vinblastine site to cause the latter compound to bind less well to tubulin. The model suggests that halichondrin B and NSC 707389 most likely form highly unstable, small aberrant tubulin polymers rather than the massive stable structures observed with vinca alkaloids and antimitotic peptides.From a structural viewpoint, halichondrin B (Fig. 1) is one of the most complex of the antitubulin compounds. Isolated from the sponges Halichondria okadai (Hirata and Uemura, 1986), Axinella species , and Lissodendoryx species (Litaudon et al., 1997), halichondrin B is a potent antimitotic agent that inhibits tubulin assembly (Bai et al., 1991). Because it inhibits the binding of radiolabeled vinblastine to tubulin in a noncompetitive manner, halichondrin B seems to bind in the "vinca domain" rather than in the same site on tubulin as the vinca alkaloids. Like most vinca domain drugs, halichondrin B inhibits both nucleotide exchange on -tubulin (Bai et al., 1991) and formation of a cross-link between -tubulin residues Cys12 and, probably, Cys211 (Ludueñ a et al., 1993). These properties have led to the assumption that the vinca domain was located near the exchangeable nucleotide site at the plus-end of the ␣-tubulin heterodimer, as defined in the electron crystallographic tubulin model (Nogales et al., 1998), in which Cys12 is only a few angstroms from the guanine residue of the exchangeable GDP. The recent success in locating the vinca site using crystals of two ␣-tubulin-colchicinoid complexes bound to a