Tubulin glycylation is an evolutionarily conserved posttranslational modification, which occurs on specific glutamic acid residues on the carboxy terminal tail domains of . and . tubulin. Tubulin glycylation is highly evolutionarly conserved from protists to mammals, and is mostly found in cells with cilia and flagella. In ciliates, the modification also occurs on non-ciliary microtubules. Previous studies showed that the glycylation domain of .tubulin has an essential role in cell survival in Tetrahymena. However, not much is known about the cellular function of this modification. The studies presented here explore the effects that a deficiency in the levels of glycylation has on ciliary and cortical structures in Tetrahymena thermophila. A lethal .tubulin glycylation domain mutation, DDDE440, was induced as a result of conjugation of genetic heterokaryons. The study revealed completely opposite effects of the same mutation on cilia and cortical organelles. The axoneme showed a lack of structural integrity, with an absence of the Btubule of peripheral doublets and the central pair. The mutants underwent consecutive and incomplete rounds of cell cycle with defective cytokinesis. Cortical microtubular structures such as longitudinal microtubules failed to undergo proper severing and appeared to impede the cleavage furrow. Cilia in the glycylation mutants were mostly affected at the early stages of their assembly with an initial delay in assembly of new cilia and complete failure in assembly of the central pair. The modification was also required for long-term maintenance of proper structure and length of cilia. Cortical microtubules such as longitudinal microtubules and basal bodies showed signs of hypertrophy. A polarized effect was seen with the anterior subcell most affected by the mutation. An arrest in nuclear and cortical growth and dissociation of a cortical microtubule associated protein occurred selectively in the anterior half of the mutant cells. The results presented here show multiple role of the tubulin glycylation domain in assembly and maintenance of microtubules. Tubulin glycylation appears to act in a context-specific manner and its function is dependent on both subcellular localization and antero-posterior cell polarity.