The plasmid pFNL1OO was created by ligation of Escherichia coli plasmid pBR328 and plasmid pFNL1O from Francisella novicida-like strain F6168. This plasmid was able to replicate and to express the genes for chloramphenicol and tetracycline resistance in both E. coli and F. tularensis. The origin of replication of pFNL1O, needed for the replication of pFNL1OO in F. tularensis, was mapped. A Sau3A-deletion derivative of pFNL1OO, designated pFNL2OO, was constructed. This plasmid could replicate only in F. tularensis and was found to be stably inherited during cultivation both on solid medium and in liquid cultures.