Comparative reverse transcription-PCR in combination with denaturing high-pressure liquid chromatography analysis was used to determine the levels of expression of soxS, marA, acrF, acrB, and acrD in multiple-antibiotic-resistant (MAR) Salmonella enterica serovar Typhimurium isolates and mutants of S. enterica serovar Typhimurium SL1344 with defined deletions. Posttherapy MAR clinical isolates had increased levels of expression of all genes except soxS. S. enterica serovar Typhimurium SL1344 Delta acrB expressed 7.9-fold more acrF than the parent strain. A strain with an acrF deletion expressed 4.6-fold more acrB. Deletion of acrB and/or acrF resulted in 2.7- to 4.3-fold more marA mRNA and 3.6- to 4.9-fold increases in the levels of expression of acrD but had a variable effect on the expression of soxS. All mutants were hypersusceptible to antibiotics, dyes, and detergents; but the MIC changes were more noticeable for SL1344 with the acrB deletion than for the mutant with the acrF disruption. These mutants had different but overlapping phenotypes, and the concentrations of ciprofloxacin accumulated by the mutants were different. These data suggest that acrB, acrF, and acrD are coordinately regulated and that their expression influences the expression of the transcriptional activators marA and soxS.