Curacin A is a thiazoline-containing lipid from the marine cyanobacterium Lyngbya majuscula. Despite being a potent inhibitor of microtubule assembly and of colchicine binding to tubulin, curacin A bears little or no structural resemblance to colchicine or to any other tubulin ligand. We investigated the interaction of curacin A with bovine brain tubulin using three different approaches. We first examined its effect on the intra-chain formation of a cross-link in β-tubulin by N,N´-ethylenebis(iodoacetamide). Formation of this cross-link, between cys 239 and cys 354 , is blocked by colchicine and its A-ring analogues as well as by various other inhibitors of colchicine binding; C-ring analogues do not inhibit its formation. Curacin A strongly inhibited formation of this cross-link. Second, we examined the effect of curacin A on the time-dependent exposure of sulfhydryl groups on tubulin as measured by alkylation with iodo[ 14 C]acetamide. Curacin A inhibited this very strongly, more so than either colchicine or podophyllotoxin. Last, we investigated the effect of curacin A on the time-dependent exposure of hydrophobic areas on the tubulin molecule. We found that curacin A had only a small effect on this process, comparable in magnitude to that of podophyllotoxin. Curacin A thus appears to have an unusual interaction with tubulin. Its binding site on tubulin is likely to overlap with that of the A-ring of colchicine.