In Gram-negative bacteria, the insertion sequence IS26 plays a major role in disseminating antibiotic resistance genes via the formation of compound transposons. Despite this, and despite being discovered over three decades ago, until recently IS26 was relatively neglected. IS26 is now known to form cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target, and a targeted conservative mechanism involving two IS in different DNA molecules. The ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end with the remainder from IS1008. These IS all have the same 14 bp terminal inverted repeats, and the Tnp26, Tnp1006 and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognise each other’s ends. In a recA- Escherichia coli strain, IS1006, IS1008 and IS1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26. However, using mixed pairs, targeted cointegration was only detected when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalysed strand transfer, followed by migration and resolution of the Holliday junction formed.