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γ-Tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of γ-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, γ-tubulin is encoded by two genes, γ-T1 and γ-T2, that produce distinct isotypes. Comparison of the γ-T1 and γ-T2 primary sequences to a Euplotesγ-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in γ-T2. Structural models of γ-T1 and γ-T2, obtained using the 3D structure of human γ-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal ‘plus end’ contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to γ-T1, the γ-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the γ-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only γ-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the γ-T1 isotype associates only with basal bodies. Our results suggest that γ-T1 and γ-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that γ-T1 and γ-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the γ-tubulin ring complex at cold temperatures.