Two genetically distinct classes of novobiocin-supersensitive mutants were isolated from Escherichia coli K-12. One class, given the phenotypic name NbsA, lies at 10 min on the E. coli chromosome. The order of the genes in this region, based on transductional analyses, is proC NbsA plsA purE. The second, NbsB, lies at 80 min. The order of the genes in this region, based on transduction analyses, is xyl cysE NbsB pyrE. Both classes of mutants show increased sensitivity to hydrophobic drugs but are different: NbsA cells tend to be more sensitive to cationic agents, whereas NbsB cells show the opposite tendency. The sole detectable biochemical alteration in NbsA strain is greater than 90% reduction in the phosphate content of the lipid A region of the lipopolysaccharide. The NbsB mutation results in lipopolysaccharide that contains primarily the stereoisomer D-glycero-D-mannoheptose, rather than L-glycero-D-mannoheptose, and which contains very little of the distal sugars. Since NbsA strains have apparently normal outer membrane proteins and total cellular phospholipids, changes solely in lipopolysaccharide can increase permeability to certain hydrophobic antibiotics. Complementation studies indicate that the NbsA marker is probably allelic with acrA. In addition, the NbsB marker is genetically and phenotypically similar to the rfaD locus of Salmonella typhimurium. For this reason, the phenotypic designations NbsA and NbsB have been changed to the genotypic designations acrA and rfaD, respectively.