In uropathogenic Escherichia coli strain 536, six pathogenicity islands (PAIs) encode key virulence factors. All PAIs except PAI IV(536) are flanked by direct repeats and four of them encode integrases responsible for their chromosomal excision. To study recombination sites used for the integration by PAI II(536) and III(536) integrases, we measured site-specific recombination between the chromosomal integration site attB, and the PAI-specific attachment site attP. We show that PAI III(536) IntB, but not IntA, mediates PAI III(536) integration. Studies of integrative recombination sites of both PAIs show that, when using a large cognate attP site (839 bp for PAI II(536) and 268 bp for PAI III(536)), PAI II(536) and III(536) attB sites could be reduced to 16 bp and 20 bp, respectively, without affecting recombination. Further reduction to 14 bp for PAI II(536) and 13 bp for PAI III(536) diminished recombination efficiency. Surprisingly, attP sites could also be reduced to 14 bp (PAI II(536)) and 20 bp (PAI III(536)). The integration host factor (IHF) and the DNA-bending HU protein do not influence PAI II(536) recombination, but IHF enhances PAI-III(536) excision and negatively affects its integration. These data suggest that PAI intasomes differ from those of lambda and P4 integrase paradigms.