Brain tubulin preparations contain an abundant type of tubulin which does not undergo the normal cycle of tyrosination-detyrosination, and whose nature is still unknown. We have used peptide sequence analysis and mass spectrometry combined with immunological procedures to show that this non-tyrosinatable tubulin has a specific primary structure. It differs from the tyrosinated isotype in that it lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit. Thus, non-tyrosinatable tubulin originates from a well-defined posttranslational modification of the tubulin primary structure which is located at the expected site of activity of tubulin tyrosine ligase. This probably accounts for the reason why it cannot be tyrosinated. The significance of this abundant brain isotubulin and the metabolic pathway involved in its formation remain to be elucidated. This should shed light on the relation between the structural diversity of the carboxy terminus of alpha-tubulin and the regulation of functional properties of microtubules.