Heterotrimeric G-proteins and their regulators are emerging as important players in modulating microtubule polymerization dynamics and in spindle force generation during cell division in C. elegans, D. melanogaster and mammals. We recently demonstrated that RGS14 is required for completion of the first mitotic division of the mouse embryo, and that it regulates microtubule organization in vivo. Here, we demonstrate that RGS14 is a microtubule-associated protein and a component of the mitotic spindle that may regulate microtubule polymerization and spindle organization. Taxol-stabilized tubulin, but not depolymerized tubulin coimmunoprecipitates with RGS14 from cell extracts. Furthermore, RGS14 copurifies with tubulin from porcine brain following multiple rounds of microtubule polymerization/depolymerization and binds directly to microtubules formed in vitro from pure tubulin (KD = 1.3 +/- 0.3 microM). Both RGS14 and Galpha(i1) in the presence of exogenous GTP promote tubulin polymerization, which is dependent on additional microtubule-associated proteins. However, preincubation of RGS14 with Galpha(i1)-GDP precludes either from promoting microtubule polymerization, suggesting that a functional GTP/GDP cycle is necessary. Finally, we show that RGS14 is a component of mitotic asters formed in vitro from HeLa cell extracts and that depletion of RGS14 from cell extracts blocks aster formation. Collectively, these results show that RGS14 is a microtubule-associated protein that may modulate microtubule dynamics and spindle formation.