The binding of two antitumour alkaloids, vinorelbine and vinflunine, to the alpha/beta-tubulin dimer has been investigated at equilibrium by nuclear magnetic resonance (NMR) spectroscopy. Tubulin stability and assembly induced by these drugs has been checked under NMR experimental conditions, and tubulin spirals were found in majority. Then, using increasing ligand concentrations, the alkaloids were titrated against tubulin. A non-specific binding of both compounds to tubulin (K(d)>10(-5)M) was characterised by broad NMR ligand signal at 4 and 30 degrees. The tubulin dimer exhibited also 2.7 (sigma: 0.3) and 2.6 (sigma: 0.6) binding sites with a K(d)<10(-5)M for vinorelbine at 4 and 30 degrees, respectively. In contrast, if the tubulin dimer exhibited 2.7 (sigma: 0.2) binding sites for vinflunine at 4 degrees, these sites were not detected at 30 degrees. This NMR study revealed for the first time the presence of specific binding sites and a clear differential affinity of vinorelbine and vinflunine to the tubulin dimer at physiological temperatures which could possibly account for their differential cytotoxicity.