A posttranslational modification was detected in the carboxyl-terminal region of axonemal tubulin from Paramecium. Tubulin carboxyl-terminal peptides were isolated and analyzed by Edman degradation sequencing, mass spectrometry, and amino acid analysis. All of the peptides, derived from both α and β tubulin subunits, were modified by polyglycylation, containing up to 34 glycyl units covalently bound to the γ carboxyl group of glutamyl residues. This modification, present in one of the most stable microtubular systems, may influence microtubule stability or axoneme function, or both.