Compounds that modulate microtubule dynamics include highly effective anticancer drugs, leading to continuing efforts to identify new agents and improve the activity of established ones. Here, we demonstrate that 3H-labeled halichondrin B (HB), a complex, sponge-derived natural product, is bound to and dissociated from tubulin rapidly at one binding site per αβ-heterodimer, with an apparent K d of 0.31 μM. We found no HB-induced aggregation of tubulin by high-performance liquid chromatography, even following column equilibration with HB. Binding of 3HHB was competitively inhibited by a newly approved clinical agent, the truncated HB analogue eribulin (apparent K i , 0.80 μM) and noncompetitively by dolastatin 10 and vincristine (apparent K i ’s, 0.35 and 5.4 μM, respectively). Our earlier studies demonstrated that HB inhibits nucleotide exchange on β-tubulin, and this, together with the results presented here, indicated the HB site is located on β-tubulin. Using molecular dynamics simulations, we determined complementary conformations of HB and β-tubulin that delineated in atomic detail binding interactions of HB with only β-tubulin, with no involvement of the α-subunit in the binding interaction. Moreover, the HB model served as a template for an eribulin binding model that furthered our understanding of the properties of eribulin as a drug. Overall, these results established a mechanistic basis for the antimitotic activity of the halichondrin class of compounds.