A native plasmid of Bacillus thuringiensis subsp. kurstaki strain YBT-1520 named pBMB9741 has been successfully cloned, sequenced, and characterized. Twelve open reading frames of at least 50 amino acids were identified. BLAST search indicated that three of them encode conserved proteins involved in conjugative mobilization, replication initiation, and transcription regulation. The orf6 located within a 2.2-kb minimal replication region was predicted to encode a replication protein. An homologous study of the orf6 product suggested that this plasmid might engage a rolling-circle replication mechanism. Unlike many other plasmids that adopt a rolling-circle model to replicate, pBMB9741 demonstrated strong segregation stability. When tested at 28 degrees C, 37 degrees C, and 42 degrees C, this plasmid maintained 100% stability in a variety of strains, including wild-type strains of B. thuringiensis and B. cereus, as well as plasmidless mutants of B. thuringiensis subsp. kurstaki and subsp. israelensis.