Determining the molecular mechanism of the neuronal Tau protein in the tubulin heterodimer assembly has been a challenge owing to the dynamic character of the complex and the large size of microtubules. We use here defined constructs comprising one or two tubulin heterodimers to characterize their association with a functional fragment of Tau, named TauF4. TauF4 binds with high affinities to the tubulin heterodimer complexes, but NMR spectroscopy shows that it remains highly dynamic, partly because of the interaction with the acidic C-terminal tails of the tubulin monomers. When bound to a single tubulin heterodimer, TauF4 is characterized by an overhanging peptide corresponding to the first of the four microtubule binding repeats of Tau. This peptide becomes immobilized in the complex with two longitudinally associated tubulin heterodimers. The longitudinal associations are favored by the fragment and contribute to Tau's functional role in microtubule assembly.