The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.