The kinetics of Taxol association to and dissociation from stabilized microtubules has been measured by competition with the reference fluorescent derivative Flutax-1 (Diaz, J. F., Strobe, R., Engelborghs, Y., Souto, A. A., and Andreu, J. M. (2000) J. Biol. Chem. 275, 26265-26276). The association rate constant at 37 degrees C is k(+) = (3.6 +/- 0.1) x 10(6) m(-1) s(-1). The reaction profile is similar to that of the first step of Flutax-1 binding, which probably corresponds to the binding of the Taxol moiety. The rate constant of the initial binding of Flutax-1 is inversely proportional to the viscosity of the solution, which is compatible with a diffusion-controlled reaction. Microtubule-associated proteins bound to the microtubule outer surface slow down the binding of Flutax-1 and Flutax-2 10-fold. The binding site is fully accessible to Flutax-2 in native cytoskeletons of PtK2 cells; the observed kinetic rates of Flutax-2 microtubule staining and de-staining are similar to the reaction rates with microtubule associated proteins-containing microtubules. The kinetic data prove that taxoids bind directly from the bulk solution to an exposed microtubule site. Several hypotheses have been analyzed to potentially reconcile these data with the location of a Taxol-binding site at the model microtubule lumen, including dynamic opening of the microtubule wall and transport from an initial Taxol-binding site at the microtubule pores.