Insulin receptor kinase phosphorylated tubulin in an insulin-dependent fashion. Two different populations of phosphotubulin were found. In tubulin dimers containing tyrosine at the carboxyl-terminal of their alpha subunit, phosphate was incorporated in that residue, and the phosphorylated protein did not assemble into polymers. In tubulin dimers lacking this tyrosine residue, phosphate was incorporated into different tyrosine residues located in other parts of the molecule, and the phosphoprotein retained its capacity to polymerize.