Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and beta gamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and beta gamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted G alpha beta gamma heterotrimer from myristoylated-G alpha and prenylated-G beta gamma, and found that the heterotrimer blocks Gi1 alpha activation of tubulin GTPase and inhibits the ability of G beta 1 gamma 2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between G alpha/G beta gamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling. (c) 2005 Elsevier Inc. All rights reserved.