A novel shuttle integration cosmid vector (pTOYAMAcos), based on pKU402, and shuttle integration vectors (pTYM18 and pTYM19) were constructed for the cloning of actinomycete DNA and its heterologous expression. These vectors contain oriT of an IncP transmissible plasmid in order to transfer genes by conjugation from Escherichia coli to actinomycetes, and they also contain int derived from actinophage φC31 in order to integrate site-specifically into the chromosomal DNA. pTOYAMAcos contains the λcos site to promote packaging of vectors containing 35-45-kb DNA fragments into λ particles. pTYM18 and pTYM19 contain kanamaycin and thiostrepton resistance genes, respectively, and have multiple cloning sites including EcoRI and HindIII sites, which are available for blue/white screening in E. coli. To demonstrate the utility of these vectors, we expressed the entire gene cluster for rebeccamycin biosynthesis from Lechevalieria aerocolonigenes using pTOYAMAcos and detected rebeccamycin production in transformed S. lividans. In addition, we demonstrated the utility of pTYM19 in a gene-disruption complementation test. L. aerocolonigenes ΔrebC strain, which is defective in rebeccamycin production because of a rebC deletion, was restored to rebeccamycin production by complemention by rebC cloned in pTYM19.