We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.