IS91 is a 1.85 kb insertion sequence originally resident in the alpha-hemolytic plasmid pSU233. The element was transposed sequentially from this plasmid to pACYC184, to R388, and to pBR322. Both cointegrates and simple insertions of the element were obtained. A detailed restriction enzyme map of the element is presented. This does not bear any relationship to the maps of previously described insertion sequences. Furthermore, hybridization between these sequences and IS91 could not be demonstrated. Deletion derivatives of IS91 were constructed which are unable to transpose. However, their transposition can be complemented in trans by wild-type elements. One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5. When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates. Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition.