The protein tubulin exhibits seven b isotypes. All except the isotype bVI are highly homologous to each other. The sequence divergence of bVI with other b isotypes is implicated in its dissimilar in vitro assembly properties. The overall goal of this project is to explore the consequences of the isotype bVI sequence divergence for ligands binding to the colchicine site. Chicken erythrocyte tubulin (CETb), which is composed solely of the aI bVI isotypes, was purified from chicken blood. Superimposition of chicken isotype bVI with the electron diffraction structure of mammalian brain tubulin-colchicine complex revealed four divergent residues within 5Å of colchicine binding site, indicating that the colchicine binding site on bVI possesses topography different from that of the other b isotypes. Colchicine did not bind to CETb even at high ligand concentrations. Podophyllotoxin binds to CETb, indicating that the colchicine A ring binding pocket on CETb is structurally compatible for the trimethoxyphenyl portion of colchicine. Surprisingly, allocolchicine, which possesses an aromatic C ring rather than the tropone ring of colchicine, binds to CETb. The kinetics of binding and fluorescence titration at 25°C revealed an apparent on rate constant and affinity constant about 23 and 28 times less than those of allocolchicine binding to bovine brain tubulin. The slow and suppressed binding of allcolchicine with CETb indicates that the C ring binding pocket on CETb may either be shrunken or incompletely accessible to the colchicine C ring.