Two variants of insertion sequence IS231, named IS231G and H, were isolated fromBacillus thuringiensissubsp.darmstadiensis73-E-10-2 (BTD2), an isolate toxic to dipteran insects, and characterized by DNA sequence analysis. They are encoded consecutively as direct repeats on anEcoRI fragment of 5.6 kilo base pairs. Direct tandem repeats of IS231 elements have not been previously reported. Both elements are closely related to other members of the IS231 family that have been isolated fromB.thuringiensisstrains toxic to lepidopteran as well as to dipteran insects. A close correlation exists between the evolutionary relationships of the IS231 sequences determined to date and the toxicity spectrum of the host cell. Probing of BTD2 DNA with a radiolabeled IS231G fragment demonstrated that IS231 elements are located on 55- and 34-MDa plasmids as well as on chromosomal DNA. Chromosomal DNA, but not plasmids, from BTD2 also hybridizes to another, unrelated insertion sequence, IS240, fromB.thuringiensissubsp.israelensis, an isolate toxic to dipteran insects. BTD2, therefore, contains IS elements once thought to reside exclusively in either dipteran- or lepidopteran-specific subspecies ofB.thuringiensis.Key words: IS231, IS240, mobile elements.