In this study,a 35-kb fragment was screened from the genome BAC library of a Bacillus thuringiensis strain with special primers.Sequence comparison of this fragment indicated that it contained a replicon of the plasmid p26.To test whether this fragment contain a replicon or not,a vector pHT304E that does not replicate in B.thuringiensis was constructed through deleting a 2.1-kb EcoRV fragment of pHT304 which containing the replication protein of B.thuringiensis.A 12-kb EcoRⅠ fragment cloned with the probe of replication vector pHT304E revealed that it was capable of supporting the replication in Bacillus strain.The replication region of p26 was still stable after 40 generations under the nonselective condition.