Previously, we have identified the association of G protein  subunit (G) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified G subunit to identify the close association of G subunit with purified brain microtubules and have shown the direct incorporation of G subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained G subunit, (2) coimmunoprecipitation demonstrated that G subunit could be coprecipitated with tubulin, (3) addition of purified G subunit into cytosolic extract for microtubule assembly caused direct incorporation of G subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous G subunit with detergent-permeabilized cells resulted in direct incorporation of G subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein  subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction.