The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiasterlin resembles most other antimitotic peptides in noncompetitively inhibiting the binding of vinblastine to tubulin (apparent K(i) value, 7.0 microM), competitively inhibiting the binding of dolastatin 10 to tubulin (apparent K(i) value, 2.0 microM), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of free drug, even at low drug concentrations. The tubulin oligomerization reaction induced by hemiasterlin was compared to the reactions induced by dolastatin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced formation of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morphology of the cryptophycin 1 aggregate consisted primarily of smaller rings (diameter about 30 nm). However, the hemiasterlin aggregate differed from the dolastatin 10 aggregate in that its formation was not associated with turbidity development, and the morphology of the hemiasterlin aggregate (as opposed to the dolastatin 10 aggregate) did not change greatly when microtubule-associated proteins were present (tight coils and pinwheels are observed with dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacification of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 degrees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory at both reaction temperatures.