The PmrA-PmrB two-component regulatory system of Salmonella enterica serovar Typhimurium is activated in vivo and plays an important role in resistance to cationic antimicrobial peptides. Resistance is partly mediated by modifications to the lipopolysaccharide. To identify new PmrA-regulated genes, microarray analysis was undertaken comparing cDNA derived from PmrA-constitutive and PmrA-null strains. A combination of RT-PCR and transcriptional analysis confirmed the inclusion of six new loci in the PmrA-PmrB regulon: STM1253, STM1269, STM4118, STM0459, STM3968 and STM4568. These loci did not affect the ability to grow in high iron conditions, the ability to modify lipid A with aminoarabinose, or virulence. STM4118, a putative phosphoethanolamine phosphotransferase, had a minor effect on polymyxin resistance, whereas the remaining genes had no role in polymyxin resistance. Although several of the identified loci lacked the consensus PmrA binding site, PmrA was demonstrated to bind the promoter of a PmrA-activated gene lacking the consensus site. A more complete definition of the PmrA-PmrB regulon will provide a better understanding of its role in host and non-host environments.