The alpha beta-tubulin heterodimer, the structural unit of microtubules, comes in many variants. There are different alpha and beta isotypes encoded by multigene families. Additional heterogeneity is generated by a set of posttranslational modifications. Detyrosination of alpha-tubulin, removal of the carboxy-terminal Glu-Tyr dipeptide of alpha-tubulin, phosphorylation of some tubulins, polyglutamylation, and polyglycylation of alpha- and beta-tubulins all involve the acidic carboxy-terminal region. We have investigated the distribution of tubulin variants in the axonemal microtubules of sea urchin sperm flagella by immunological procedures and by direct sequence and mass spectrometric analysis of the carboxy-terminal peptides. The A and B tubules that comprise the nine outer doublets differ strongly in tubulin variants. A tubules contain over 95% unmodified, tyrosinated alpha beta-tubulin. In B tubules, alpha-tubulin is approximately 65% detyrosinated and both alpha- and beta-tubulin are 40-45% polyglycylated. These results show a segregation of tubulin variants between two different axonemal structures and raise the possibility that posttranslational modifications of tubulins reflect or specify structurally and functionally distinct microtubules.