p190RhoGAP (p190), a Rho family GTPase-activating protein, regulates actin stress fiber dynamics via hydrolysis of Rho-GTP. Recent data suggest that p190 also regulates cell proliferation. To gain insights into the cellular process(es) affected by p190, we altered its levels by conditional or transient overexpression. Overexpression of p190 resulted in a multinucleated phenotype that was dependent on the GTPase-activating protein domain. Confocal immunofluorescence microscopy revealed that both endogenous and exogenous p190 localized to the newly forming and contracting cleavage furrow of dividing cells. However, overexpression of p190 resulted in abnormal positioning of the furrow specification site and unequal daughter cell partitioning, as well as faulty furrow contraction and multinucleation. Furthermore, levels of endogenous p190 protein were transiently decreased in late mitosis via an ubiquitin-mediated degradation process that required the NH2-terminal GTP-binding region of p190. These results suggest that a cell cycle-regulated reduction in endogenous p190 levels is linked to completion of cytokinesis and generation of viable cell progeny.