Chinese hamster ovary cells grown in monolayer culture and exposed to cytochalasin B were enucleated by centrifugation. Thereafter, the karyoplasts (the nucleated parts obtained from the bottoms of the centrifuge tubes) and the cytoplasts (the enucleated cytoplasmic parts attached to the coverslips) were allowed to recover and subsequently were examined by scanning and transmission electron microscopy. Microscopy of thin sections revealed that the karyoplasts, limited by an intact plasma membrane, contain an intact nucleus surrounded by a layer of cytoplasm that includes ribosomes, mitochondria, and fragments of the endoplasmic reticulum, but no centrioles or microtubules. The cytoplasts, similarly examined, appear to contain all cytoplasmic organelles and systems, including centrioles and microtubules. The karyoplasts, when replated in fresh medium adhere to the substrate but remain essentially spherical and are incapable of motility. They disintegrate in about 72 hr. The cytoplasts, under identical conditions, recover a shape similar to that of the whole Chinese hamster ovary cell and display some motility. They generally survive not more than 48 hr. It appears that this enucleation procedure consistently separates the nucleus and limited cytoplasm from the centrosphere and microtubule-containing cytoplasts and, furthermore, that the formdetermining and motility mechanisms reside in the cytoplast and function without nuclear participation for the short period of viability.