During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell^,,, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody,. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures,, is known to be essential^,,. Protein kinases are also thought to participate in cytokinesis^,,,,. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK and ROK^,,, which regulate myosin-based contractility^,,,. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.