IS493 is an insertion sequence isolated from Streptomyces lividans by a method designed to 'trap' transposable elements. IS493 was converted to functional transposons by cloning antibiotic-resistance-encoding genes between ORF-A and ORF-B of IS493 or near the left-end inverted repeat of the element. Tn5096 transposed relatively randomly in several Streptomyces species. Tn5096 can be introduced into streptomycetes on temperature-sensitive vectors by protoplast transformation, FP43-mediated transduction, or by conjugation from Escherichia coli. We have shown that additional genes can be inserted in Tn5096 without disrupting transposition, and that Tn5096 insertions in a tylosin (Ty)-producing strain of Streptomyces fradiae frequently cause no deleterious effects on Ty production. A promoter probe transposon, Tn5099, containing a promoterless xylE gene, transposed in Streptomyces griseofuscus and S. fradiae, and transcriptional fusions were readily identified.