We have recently cloned a cDNA for a full-length form of MgcRacGAP. Here we show using anti-MgcRacGAP antibodies that, unlike other known GAPs for Rho family, MgcRacGAP localized to the nucleus in interphase, accumulated to the mitotic spindle in metaphase, and was condensed in the midbody during cytokinesis. Overexpression of an N-terminal deletion mutant resulted in the production of multinucleated cells in HeLa cells. This mutant lost the ability to localize in the mitotic spindle and midbody. MgcRacGAP was also found to bind alpha-, beta-, and gamma-tubulins through its N-terminal myosin-like domain. These results indicate that MgcRacGAP dynamically moves during cell cycle progression probably through binding to tubulins and plays critical roles in cytokinesis. Furthermore, using a GAP-inactive mutant, we have shown that the GAP activity of MgcRacGAP is required for cytokinesis, suggesting that inactivation of the Rho family of GTPases may be required for normal progression of cytokinesis.