Techniques of individual cell selection and precise ultramicrotomy have been employed to demonstrate that the contractile ring of cleaving HeLa cells is a transitory cytoplasmic organelle of distinctive fine structure and location. The contractile ring is an uninterrupted annulus encircling the equator of dividing cells exactly where the cleavage furrow forms. It is about 10 microns wide, up to 0.2 microns in thickness, and is composed exclusively of circumferentially aligned thin filaments 40–70 A in diameter. Contractile ring filaments appear to be associated with the overlying plasma membrane. Controlled experiments with a mold metabolite (cytochalasin B) reveals that within a few minutes the drug abolishes the ability of HeLa cells to undergo cytokinesis. Cytochalasin B seems to decompose the contractile ring. It has no other clearly identifiable effects on other cell structures, notably the mitotic apparatus. Cytochalasin B is the only drug known which selectively inhibits cytokinesis in animal cells. In conclusion, the contractile ring is the most likely organelle responsible for cytokinesis in HeLa cells. Similar organelles probably occur in all cleaving animal cells. Successful cleavage depends upon the structural and functional integrity of the contractile ring.