The domain structure of the alpha and beta tubulin monomers and their association to the noncovalent dimer has been investigated by several independent approaches: binding of the polarity sensitive dye nile red, and equilibrium centrifugation with a newly developed short column method. Using steady state and time resolved fluorescence we find that binding of nile red to tubulin enhances and blue shifts fluorescence emission to about 623 run with a "shoulder" around 665 nm. Binding is reversible and saturable, has lifetimes of 4.5 ns and 0.6 ns fluorescence in Mes buffer associated with the 623 nm peak and the 665 nm shoulder, and has rotational correlation times of >50 ns for the 4.5 ns lifetime and 0.3 ns for the 0.6 ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623 nm component. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution and permits estimation of a subunit dissociation constant of 1 microM. Because of the ability of tubulin, a rapid, short-column equilibrium centrifugation method was developed in which equilibrium is attained in a few hours. Monomer-dimer distribution analysis yields a dissociation constant of 12xlO(-7)M at 5 degrees centigrade. Thermodynamic analysis show that dimerization is entropically driven.