IS91 is an insertion sequence of 1830 bp, which was isolated from a hemolysin plasmid of Escherichia coli. It has inverted repeats of only 8 bp and, unusually, shows an absolute target site specificity [GAAC/CAAG] (1,2). IS97 is related to IS807 from Pseudomonas syringae (3). In particular, their transposases are 35% identical proteins of 426 and 410 amino acids respectively (2,3). Apart from this, IS91 is unrelated to other presently known IS elements. Figure 1 shows the four conserved motifs between the IS91/IS8O1 transposases and a family of five replication proteins of plasmids pUBHO, pLABlOOO, pLPl, pFTB14 and pC194 from gram-positive bacteria (4, 5). Motif I shows a cysteine-rich sequence which is a potential metal-binding domain. Motif II includes an H.H.H motif. This motif is also present in a family of nickases/relaxases involved in oriT nicking for conjugal DNA transfer in a variety of plasmids, and in the VirD2 proteins responsible for nicking of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens (7). Motif IH contains the Y ... Y..K sequence. This motif is also found in the gpA protein of the ssDNA phage 0X-174 (Figure 1) and related phages, and contains the two tyrosines forming the catalytic site of these topoisomerases (8). Motif IV contains the invariant sequence R ... Y.G; the Rep protein of pC194 finishes before this motif. The pUBl 10 and related Rep proteins produce a single-strand nick in a specific site of the plasmid (the leading strand origin of replication), and this nick results in single-strand replication by the rolling circle mechanism (4, 6). Their similarity to the transposases of IS91/IS801 suggests that transposition of these elements also involves single-strand nicking by the transposases, followed by single-stranded rolling-circle replication of the transposon. This will be in contrast to any of the mechanisms of transposition that have been shown or suggested for other elements (9). Finally, it can be speculated that the IS97 transposase appeared by evolution of a plasmid Rep protein, suggesting a mechanism to explain the origin of transposable elements.