The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of α-tubulin can be posttranslationally labeled after incubation of cells with [^3H]acetate or [^3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the γ-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of α-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.