When subcloned into low-copy-number expression vectors, rumAB, encoding polV (RumA' B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polV is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polV . Indeed, we recently discovered that SetR , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroS , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetR and CroS , with the greatest reduction of RumA protein levels observed in the absence of SetR and the presence of CroS . Under these conditions, CroS completely abolishes the high levels of mutagenesis promoted by polV expressed from low-copy-number plasmids. Furthermore, deletion of croS on the native R391 results in a dramatic increase in mutagenesis, indicating that CroS plays a major role in suppressing polV mutagenesis in vivo. Inactivating mutations in CroS therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391.