It has been reported that zona-free mammalian oocytes treated with a cytoskeleton inhibitor are separated into several fragments with or without the chromatin by centrifugation. The utilization of these fragments for nuclear or cytoplasmic transfer would increase the efficiency of preparing recipient or donor cytoplasts. In the present study, the separation of mouse oocyte fragments by centrifugation was examined, and the characteristics of these fragments were evaluated. Zona-free mouse oocytes were treated with cytochalasin D, loaded individually onto a discontinuous Percoll gradient in a capillary tube, and centrifuged at 9,500 g for 1–3 min. Oocyte fragments were recovered and examined for the distributions of chromatin, lipids, and mitochondria. After centrifugation for 1 min, 57% of the oocytes treated with cytochalasin D at 4–8°C were divided into pairs of small (diameter: 27.6 ± 7.2 μm) and large (diameter: 73.5 ± 2.4 μm) fragments. Most of the large fragments did not contain any chromatin (92–100%) and showed strong and homogeneous mitochondrial fluorescence. The lipids in the oocytes moved to the centripetal side, and a part of the lipids might have been expelled to the small fragments. The present results suggest that the large fragments derived from the oocytes by centrifugation may be available as a potential source of recipient cytoplasts that are necessary for the nuclear transfer technique.