In cold poikilotherm organisms, microtubule assembly is promoted at temperatures below 4ยฐC and cold-induced depolymerization is prevented. On the basis of the results of investigations on cold-adapted fishes, the property of cold adaptation is ascribed to intrinsic characteristics of the tubulins. To fully understand cold adaptation, we studied the tubulins of Euplotes focardii, an Antarctic ciliated protozoan adapted to temperatures ranging from ฯช2 to ฯฉ4ยฐC. In this organism, we had previously sequenced one โค-tubulin gene and, then identified three other genes (denoted as โค-T1, โค-T2, โค-T3 and โค-T4). Here we report that the amino acid sequence of the carboxy-terminal domain predicted from the โค-T3 gene (apparently the most expressed of the gene family) contains six modifications (five substitutions and one insertion) of conserved residues, unique with respect to all the other known โค-tubulin sequences. These modifications can change the structural conformation of the carboxy-terminal domain. Furthermore, in the variable terminal end of that domain, a consensus sequence for a phosphorylation site is present, and the residue Glu-438, the most frequent site for polyglutamylation in โค-tubulin, is substituted by Asp. Starting from these observations, we showed that in E. focardii only โฃ-tubulin is polyglutamylated, while โค-tubulin undergoes phosphorylation. Polyglutamylated microtubules appear to colocalize with cilia and microtubular bundles, all structures in which microtubules undergo a sliding process. This finding supports the idea that โฃ-tubulin polyglutamylation is involved in the interaction between tubulin and motor microtubule-associated proteins. Phosphorylation, usually a rare posttranslational modification of โค-tubulin, which is found extensively distributed in the โค-tubulin of this cold-adapted organism, may play a determinant role in the dynamic of polymerization and depolymerization at low temperatures.