Development of a new and effecient tuberculosis vaccine is very important since the efficacy of the only available vaccine against tuberculosis, BCG, is variable among races and different ages. Attempts to develop attenuated vaccines by disrupting virulence gene(s) specifically in Mycobacterium tuberculosis are now actively being tried after the release of whole genome sequence of M. tuberculosis in 1998. However, disruption of specific genes in M. tuberculosis is still very difficult due to the lack of effective gene knock-out system(s) in mycobacteria. In this study, we developed a novel method to delete specific genes in both Escherichia coli and mycobacteria. This knock-out system is operated by a sequence-specific recombinase FLP and its recognition sequence FRT (FLP/FRT system). Two shuttle vectors (an FLP expressing vector and a gene targeting vector) between Escherichia coli and Mycobacteria were developed. The gene targeting vector contains a kanamycin resistance gene (KmR) flanked by two neighboring genes and two FRTs (FLPrecognition targets). We applied this system to knock-out the rhamnose biosynthetic gene rmlD of Escherichia coli. The upstream and downstream genes of rmlD, rmlB and rmlA, were cloned into the gene targeting vector. After and allelic exchange of E. coli chromosomal rmlB, rmlD, rmlA with vectoral rmlB, FRT-KmR-FRT, rmlA by homologous recombination, FLP-expressing plasmid was introduced to induce the excision of KmR cassette remaining one FRT sequence between rmlB and rmlA. We also demonstrated our shuttle vector could disrupt a target gene (kanamycin resistance gene) in M. smegmatis. These results suggest that our gene knock-out system can be used for the development of an attenuated tuberculosis vaccines and for the functional genomic study of mycobacteria.