The intensity of coumarin-30 fluorescence emission increased by an order of magnitude when an excess of tubulin was added to coumarin-30 solution in vitro (Fig. ). This enabled us to measure the apparent dissociation constant of coumarin-30, Kd = 3.2 ± 0.2 μM, by titrating the concentration of tubulin to a fixed concentration of coumarin-30 (Fig. ). When the solutions of coumarin-30 and tubulin were supplemented with 100 μM colchicine, the fluorescence enhancement was suppressed (Fig. ), suggesting that coumarin-30 competed with colchicine for the same binding site on tubulin. No suppression of fluorescence enhancement was observed upon supplementing the solution with 100 μM pironetin, a ligand having a distinct binding site on tubulin (Fig. ).