Cytokinesis follows nuclear division and generates two distinct daughter cells, each replete with a full complement of the genome and cytoplasmic organelles. Members of the Rho family of GTPases are crucial regulators of this process in a wide variety of species. In many cell types, cytokinesis is mediated by a discretely localized contractile ring that is rich in actin and myosin. In this article (which is part of the Cytokinesis series), we review recent studies in animal cells that have shown that local assembly of the contractile ring is mediated by a discrete pool of GTP-bound, active RhoA. Advances in detecting the active pool of RhoA have allowed insights into the mechanisms and the molecules that promote the accumulation of active RhoA at the correct time and place in the cell.