The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like integrase was required for their excision. PAI III(536) carries two integrase genes, intA, encoding an SfX-like integrase, and intB, coding for an integrase with weak similarity to P4-like integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like integrase gene but additional deletion of the PAI II(536)-specific integrase gene was required. Therefore, although all mediated by P4-like integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between integrases of different PAIs and shows that this cross-talk is unidirectional.