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The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets. We performed an in silico analysis of possible targets of the PmrAB system. Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes. To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure. As well as the known targets, we detected additional targets with unknown functions. Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ). Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements. We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally. We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.