Rat brain tubulin purified by colchicine-agarose affinity chromatography contains protein kinase activity. The kinase activity was separated completely from tubulin by chromatography on casein columns and was not subsequently retained by colchicine affinity columns. Protein kinase activity associated with purified tubulin did not correlate with the total content of protein kinase activity in brain homogenates, since microtubules isolated from 48,000 g fetal brain supernatants contained twice as much protein kinase activity than adult microtubules, although the total protein kinase activity was twice as high in the 48,000 g adult supernatant. The protein kinase of tubulin preparations, while corresponding to a different molecule than tubulin, was probably not simply the result of contamination. These observations were interpreted in terms of specific associations between protein kinase and tubulin complexes. The protein kinase-tubulin association may be an important determinant in the regulation of tubulin function. Fetal tubulin polymerized twice as well as adult tubulin in the absence of glycerol at the same tubulin concentration. The preferred substrate for the protein kinase either in vivo or in vitro (pH 7.4, 37.degree. C) was a specific high-molecular-weight protein, distinct from tubulin, which copurified with tubulin through different kinds of isolation procedures (i.e., colchicine affinity chromatography and (NH4)2SO4 precipitation followed by diethylaminoethyl-cellulose chromatography). The tubulin-associated protein kinase was completely dependent on cyclic[c]AMP (Km = 10-7 M), as demonstrated by the complete suppression of activity upon addition of the protein kinase modulator, a well-known specific inhibitor of cAMP-dependent protein kinases.