We have used fluorescence correlation spectroscopy to analyze the interaction of GTP-tubulin with rhodamine-labeled RB3, a neural protein of the stathmin family, and to determine the kinetic pathway of the association process. RB3 displayed slow association-dissociation kinetics with tubulin depending on the square of the tubulin concentration. The values of the apparent association and dissociation rate constants of the complex of two tubulin dimers and RB3 are determined to be (3.52 +/- 0.14) x 10(-3) muM(-2)/s and (1.9 +/- 0.6) X 10(-3) s(-1) respectively. The value of the equilibrium dissociation constant for the first tubulin-RB3 interaction is estimated to be greater than or equal to7 muM at 20degreesC. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.