This paper reports the results of a study about the segregational stability of a series of pNK289-derived plasmids. The segregational stability in B. subtilis AS1. 1176 was found to be different among these same originated plasmids. The difference in segregational stability was shown not to be related to the sizes or replication patterns of the plasmids, but to have some relation to their copy numbers. As the unstable plasmid pNK219 can be miaintained stably in B. subtilis BD 224, it can be inferred that the host inheritable background could affect the segregational stability of plasmid. These results not only have laid a foundation for finding out some genes related to the stability of pNK289-derived plasmids, but also provided theoretical basis for constructing stable recombinant plasmid in Bacillus.