This study was investigated as a step for the purpose of successful introduction of cytoplasmic inherited characters between the different plants. Cytoplasts were separated from the protoplasts of CMS(cytoplasmic male sterility) line such as MS Burley 21 which carried from Nicotiana megalosiphon. The cytoplasts were fused to protoplasts derived from Nicotiana tabacum Br 64 with PEG(polyethylene g1yco1). The cytoplasts were separated by density gradient centrifugation. Efficient separation of cytoplasts depended on the difference of specific density of gradient solution. However, the iso-osmolality of gradient solution was not important to separate the cytoplasts. The cells for a cybrid were fused with 50% concentration of PEG.