The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to λ phage. However, contrary to the λ superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Φ24B. Here, the Φ24B integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Φ24B integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Φ24B gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Φ24B E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Φ24B to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.