In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly ; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures . MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations . We have now found that the activity copurifies with the projection portion of MAP 2 itself . Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel-filtration chromatography on Bio-Gel A15m or ion-exchange chromatography on DEAE-Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface . The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on Bio-Gel A-15m . Three protein species (Mr = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (Mr = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP-dependent protein kinase . The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited >95% by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle . It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPS, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22% of the total brain cytosolic protein kinase activity . The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible .