Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.