Colchicine, which is known to influence tubulin function, was used to delineate a possible role of tubulin in signal transduction in the rat striatal membranes. Low Km GTPase activity (EC 3.6.1.-) was assayed in 10 micrograms membrane protein using [gamma-32P]GTP at 37 degrees in an ATP-regenerating buffer containing 1 microM unlabeled GTP. At 10 and 100 microM, colchicine inhibited the GTPase activity by 18% and 40%, respectively. Colchicine (100 microM) inhibited the enzymatic activity by 30-40% at all the time points the reaction was monitored. Acetylcholine (ACh) stimulated the low Km GTPase activity in a concentration-dependent manner, by up to 57%. ACh-stimulated activity was accepted as reflecting GTP hydrolysis catalyzed by receptor-coupled transducer G proteins. In the presence of 100 microM colchicine, the ability of ACh to stimulate G protein GTPase activity was inhibited. For example, at 10 microM ACh the enzyme activity was stimulated up to 52%; in the presence of 100 microM colchicine, 10 microM ACh stimulated the activity by only up to 33%. These results suggest that colchicine disrupts ACh receptor-G protein coupling as a result of its interaction with tubulin or G protein(s) or both.