Halichondrin B is a polyether macrolide of marine origin which binds to tubulin and inhibits microtubule assembly in vitro and in vivo. As is the case with phomopsin A and dolastatin 10, halichondrin B is a non-competitive inhibitor of vinblastine binding to tubulin. Analogous to maytansine, which by contrast is a competitive inhibitor of vinblastine binding, halichondrin B has no effect on colchicine binding, which is greatly stabilized by phomopsin A and dolastatin 10, but not by maytansine. We have previously developed assays which allow sensitive discrimination among the interactions of various ligands with tubulin, and examined the effects of ligands on the reactivity of tubulin sulfhydryl groups and the exposure of hydrophobic areas on the surface of the tubulin molecule. To classify the nature of the interaction between halichondrin B and tubulin, in this study we examined the effects of halichondrin B and its closely related analogue, homohalichondrin B, by these assays. We found that: (1) halichondrin B and homohalichondrin B both inhibited formation of an intra-chain cross-link between two sulfhydryl groups in beta-tubulin, as do phomopsin A, dolastatin 10, maytansine, and vinblastine; (2) halichondrin B resembles maytansine in that it had no effect on alkylation of tubulin sulfhydryl groups by iodoacetamide, unlike phomopsin A, dolastatin 10 and vinblastine, all of which inhibit alkylation; (3) halichondrin B differs from other anti-mitotic drugs in that it enhanced exposure of hydrophobic areas on tubulin; (4) homohalichondrin B, like maytansine and in contrast to phomopsin A, dolastatin 10 and vinblastine, had no effect on exposure of hydrophobic areas; and (5) homohalichondrin B, contrary to maytansine, inhibited alkylation of tubulin sulfhydryl groups in the presence of GTP and MgCl2. In their interactions with the tubulin molecule, halichondrin B and homohalichondrin B appear to have unique conformational effects which differ from those of other drugs and also from the effects of each other as well.