The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.