Acquisition of new prophages that are able to increase the bacterial fitness by the lysogenic conversion is believed to be an important strategy of bacterial adaptation to the changing environment. However, in contrast to the factors determining the range of bacteriophage lytic activity, little is known about the factors that define the lysogenization host range. Bacteriophage phi24B is the paradigmal model of Stx-converting phages, encoding the toxins of the Shiga-toxigenic E. coli (STEC). This virus has been shown to lysogenize a wide range of E. coli strains that is much broader than the range of the strains supporting its lytic growth. Therefore, phages produced by the STEC population colonizing the small or large intestine are potentially able to lysogenize symbiotic E. coli in the hindgut, and these secondary lysogens may contribute to the overall patient toxic load and to lead to the emergence of new pathogenic STEC strains. We demonstrate, however, that O antigen effectively limit the lysogenization of the wild E. coli strains by phi24B phage. The lysogens are formed from the spontaneous rough mutants and therefore have increased sensitivity to other bacteriophages and to the bactericidal activity of the serum if compared to their respective parental strains.