The gene encoding the general stress transcription factor ς^B in the gram-positive bacterium Listeria monocytogenes was isolated with degenerate PCR primers followed by inverse PCR amplification. Evidence for gene identification includes the following: (i) phylogenetic analyses of reported amino acid sequences for ς^B and the closely related ς^Fproteins grouped L. monocytogenes ς^B in the same cluster with the ς^B proteins from Bacillus subtilis and Staphylococcus aureus, (ii) the gene order in the 2,668-bp portion of the L. monocytogenes sigBoperon is rsbU-rsbV-rsbW-sigB-rsbX and is therefore identical to the order of the last five genes of the B. subtilis sigB operon, and (iii) an L. monocytogenesς^B mutant had reduced resistance to acid stress in comparison with its isogenic parent strain. The sigB mutant was further characterized in mouse models of listeriosis by determining recovery rates of the wild-type and mutant strains from livers and spleens following intragastric or intraperitoneal infection. Our results suggest that ς^B-directed genes do not appear to be essential for the spread of L. monocytogenes to mouse liver or spleen at 2 and 4 days following intragastric or intraperitoneal infection.