Tubulin was found to bind to a glutathione S-transferase fusion protein containing the carboxy-terminal domain of GTP-binding-protein-coupled receptor kinase 2 (GRK2) (residues 467−689), which is known to contain a pleckstrin homology site and to bind GTP-binding protein βγ subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein βγ subunits, indicating that tubulin and βγ subunits bind GRK2 independently. Western-blotting analysis with anti-GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization-depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP-binding-protein-coupled receptors and that tubulin is a poor substrate for most kinases. GRK2 did not phosphorylate microtubule-associated proteins (MAPs), under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The Km for tubulin was estimated to be 3 μM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by βγ subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2.