ABSTRACT.    αβ-tubulin heterodimers of psychrophilic eukaryotes can polymerize into microtubules at 4 °C, a temperature at which microtubules from mesophiles disassemble. Microtubules are involved in many essential processes of the eukaryotic cell, as division, motility, intracellular transport, and maintenance of cell architectures. To contribute to the understanding of the molecular mechanisms responsible for microtubule cold-stability, we are studying the tubulin genes of Euplotes focardii, a ciliate endemic to the Antarctic coastal sea water. This unicellular eukaryote is exceptionally rich in microtubules and it shows strictly psychrophilic features such as optimal growth rate at 4-5 °C. By turbidimetric measurement and electron microscopy, we show that MAP-free tubulin dimers from E. focardii are able to polymerize in vitro at 4 °C. In E. focardii, the β-tubulin gene family is composed by four genes, encoding four isotypes. When compared with β-tubulin of mesophilic ciliates, the four E. focardii β-tubulin isotypes contain several unique residue substitutions. Mapping these substitutions onto the three-dimensional structures of the tubulin dimers, we find that they are localized to surfaces that are involved in lateral and longitudinal interactions, and in a region that forms part of the nucleotide binding site. Two hydrophobic substitutions common to all isotypes, C232V and V268I, may stabilize the assembly-competent conformation of tubulin at cold temperatures. Finally, one of the most divergent isotype, the β-T3, attains its native state by a non-canonical pathway, i.e. it needs unidentified cofactors in addition to the molecular chaperons involved in the folding of more conserved β-tubulin isotypes. This behavior may be a consequence of peculiar dynamic properties of the β-T3 isotype, since it specifically localizes in microtubules with a low turnover in E. focardii and in transfected mammalian cells.