The biosynthetic gene cluster for lariatins A and B, anti-mycobacterial peptide antibiotics with a unique "lasso" structure, was cloned from Gram-positive bacterium Rhodococcus jostii K01-B0171. Random transposition mutagenesis using IS1415 derivative was carried out to identify a chromosomal locus involved in lariatin biosynthesis and six independent lariatin non-producing variants were obtained. Arbitrary PCR revealed that one insertion was located near the region involved in lariatin biosynthesis. Using the lariatin gene as a probe, a genomic library of R. jostii K01-B0171 was screened by colony hybridization, and two clones were obtained. Sequence analysis of these clones revealed that the gene cluster for lariatin biosynthesis spanning about 4.5 kb consisted of five open reading frames (larA to larE). We proposed that the linear precursor LarA is processed by LarB, LarC, and LarD, and the mature lariatin is exported by LarE.