How myosin II localizes to the cleavage furrow of dividing cells is largely unknown. We show here that a 283-residue protein, assembly domain (AD)1, corresponding to the AD in the tail of Dictyostelium myosin II assembles into bundles of long tubules when expressed in myosin II-null cells and localizes to the cleavage furrow of dividing cells. AD1 mutants that do not polymerize in vitro do not go to the cleavage furrow in vivo. An assembly-competent polypeptide corresponding to the C-terminal 256 residues of Acanthamoeba myosin II also goes to the cleavage furrow of Dictyostelium myosin II-null cells. When overexpressed in wild-type cells, AD1 colocalizes with endogenous myosin II (possibly as a copolymer) in interphase, motile, and dividing cells and under caps of Con A receptors but has no effect on myosin II-dependent functions. These results suggest that neither a specific sequence, other than that required for polymerization, nor interaction with other proteins is required for localization of myosin II to the cleavage furrow.