Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.