We have investigated the solution conformation of the functionally relevant C-terminal extremes of α- and β-tubulin, employing the model recombinant peptides RL52α3 and RL33β6, which correspond to the amino acid sequences 404–451(end) and 394–445(end) of the main vertebrate isotypes of α- and β-tubulin, respectively, and synthetic peptides with the α-tubulin(430–443) and β-tubulin(412–431) internal sequences. α(404–451) and β(394–445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of β(394–445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409–Glu410 and Tyr422–Gln423–Gln424, defining the proteolysis resistant segment 410–422, which corresponds to the central part of the predicted β-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in α(404–451) and β(394–445). A substantial part of the helicity of β(394–445) was found to be in the CD spectrum of the shorter peptide β(412–431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational CαH shifts) in 30% TFE permitted to conclude that about 25% of α(404–451) and 40% of β(394–451) form well-defined helices encompassing residues 418–432 and 408–431, respectively, flanked by disordered N- and C-segments. The side chains of β(394–451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both α and β chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of α(404–451) is the same as that described in the electron crystallographic model structure of αβ-tubulin, while in β(394–451) it extends for nine residues more, supporting the possibility of a functional coil → helix transition at the C-terminus of β-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.