Two-dimensional electrophoretic analysis of crude microtubule preparations from the rat brain revealed the presence of three polypeptides in positions corresponding to those of the isovariants of purified rat brain creatine kinase (CK-BB). By the use of [gamma-32P]ATP, the two more acidic forms of these polypeptides were shown to be phosphorylated. Their identity as phosphorylated forms of CK-BB was established by using various peptide mapping techniques. Thus CK-BB is a phosphoprotein and its isoelectric variation may be attributed to phosphorylation.