Deletion of perR in Listeria monocytogenes results in a small-colony phenotype (ΔperRsm) that is slow growing and exhibits increased sensitivity to H2O2. At a relatively high frequency, large-colony variants (ΔperRlg) arise, which are more resistant to H2O2 than the wild-type and ultimately dominate the culture. Transcriptional analysis revealed that the kat gene (catalase) is up-regulated in both types of mutants and that the highest level is apparent in ΔperRsm mutants, demonstrating PerR regulation of this gene. Overexpression of the catalase gene in the wild-type background resulted in a slower-growing strain with a smaller colony size similar to that of ΔperRsm. By combining a bioinformatic approach with experimental evidence, other PerR-regulated genes were identified, including fur, lmo0641, fri, lmo1604, hemA, and trxB. The transcriptional profile of these genes in both mutant backgrounds was similar to that of catalase in that a higher level of expression was observed in ΔperRsm than in the wild type or ΔperRlg. Murine studies revealed that the virulence potential of the ΔperRsm mutant is substantially reduced compared to that of the wild-type and ΔperRlg strains. Collectively, the data demonstrate that the ΔperRsm mutant represents the true phenotype associated with the absence of PerR, which is linked to overexpression of regulated genes that negatively affect bacterial homeostasis both in vitro and in vivo. A subsequent secondary mutation occurred at a high frequency, which resulted in phenotypic reversion to a large-colony phenotype with increased fitness that may have obstructed the analysis of the role of PerR in the physiology of the bacterial cell.