Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli. In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G(2)-M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential "cargo" genes or "morons" and therefore play a crucial role in the generation of genetic diversity within ExPEC.