Modification of lipid A with the 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety is required for resistance to polymyxin and cationic antimicrobial peptides in Escherichia coli and Salmonella typhimurium. An operon of seven genes (designated pmrHFIJKLM in S. typhimurium), which is regulated by the PmrA transcription factor and is also present in E. coli, is necessary for the maintenance of polymyxin resistance. We previously elucidated the roles of pmrHFIJK in the biosynthesis and attachment of L-Ara4N to lipid A, and renamed these genes arnBCADT, respectively. We now propose functions for the last two genes of the operon, pmrL and pmrM. Chromosomal inactivation of each of these genes in an E. coli pmrA constitutive parent switched its phenotype from polymyxin-resistant to polymyxin-sensitive. Lipid A was no longer modified with L-Ara4N, even though the levels of the lipid-linked donor of the L-Ara4N moiety, undecaprenyl phosphate-alpha-L-Ara4N, were not reduced in the mutants. However, the undecaprenyl phosphate-alpha-L-Ara4N present in the mutants was less concentrated on the periplasmic surface of the inner membrane, as judged by 4- to 5-fold reduced labeling with the inner membrane-impermeable amine reagent N-hydroxysulfosuccinimidobiotin. In an arnT mutant of the same pmrA constitutive parent, which lacks the enzyme that transfers the L-Ara4N unit to lipid A but retains the same high levels of undecaprenyl phosphate-alpha-L-Ara4N as the parent, N-hydroxysulfosuccinimidobiotin labeling was not reduced. These results implicate pmrL and pmrM, but not arnT, in transporting undecaprenyl phosphate-alpha-L-Ara4N across the inner membrane. PmrM and PmrL, now renamed ArnE and ArnF because of their involvement in L-Ara4N modification of lipid A, may be subunits of an undecaprenyl phosphate-alpha-L-Ara4N flippase.