Technical Abstract: Stationary phase sigma-factor (RpoS) regulates many of the cell’s responses towards environmental stress conditions such as heat, acid and alkali-shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of enterohemorrhagic E. coli strains from food-borne outbreaks with reference to their RpoS-mediated stress responses. E. coli strains belonging to serotypes O157:H7, O111:H12 and O26:H11 exhibited polymorphism for the two phenotypes widely used to monitor rpoS mutations: heat-tolerance and glycogen synthesis, even though they synthesized the oxidative acid-resistance system by the rpoS-dependent pathway. Both of the phenotypes were restored upon introduction of a wild-type rpoS on a low-copy number plasmid. During the transition from a mildly-acidic growth condition to alkaline stress, the cells relied heavily on rpoS functionality. Some strains were able to overcome negative-regulation by the RpoS and induced higher Beta-galactosidase activity without compromising their acid-resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the various strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was not evident in the Caco-2 cells adherence assays. These results show that some pathogenic E. coli strains can modulate their rpoS-dependent cellular responses without affecting their acid-tolerance capability. Such adaptation processes may have significant impact on a pathogen’s survival in food processing environments as well in the host’s stomach and intestine.