ABSTRACTContributions of the alternative sigma factor σBtoListeria monocytogenesinfection were investigated using strains bearing null mutations insigB,prfA, orinlAor in selectedinlAorprfApromoter regions. The ΔP4inlAstrain, which has a deletion in the σB-dependent P4inlApromoter, and the ΔsigBstrain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σBdependent P2prfApromoter region did not differ from that of the wild type. The virulence of the ΔsigBand ΔP4inlAstrains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigBstrain showing greater attenuation, while the virulence capacity of the ΔP2prfAstrain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigBmutation does not result from loss of σB-dependentprfAtranscription. Our results show that σB-dependent activation ofinlAis important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition toinlAappear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigBstrain was not attenuated in intravenously infected guinea pigs. We conclude that (i)L. monocytogenesσBplays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects oninlAtranscription but not onprfAtranscription, and (iii) σBplays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.