The bactericidal effect of polymyxin B upon two Pseudomonas aeruginosa strains and two Escherichia coli strains was studied as a function of the concentration of the divalent cations Ca2+ and Mg2+. Lipid spherules in aqueous suspension (liposomes) were prepared from the bacterial phospholipids and were examined for the release of trapped glucose induced by polymyxin B under similar conditions of divalent cation concentration. In contrast to the bacteria, which were protected markedly from the bactericidal action of polymyxin B by Ca2+ or Mg2+ at concentrations of 2 × 10−3m or less, neither cation had any protective effect on bacterial liposomes at concentrations up to 2 × 10−2m. These observations suggest that divalent cations antagonize the bactericidal effect of polymyxin B indirectly through interaction with the cell wall rather than at the primary membrane locus of action. The interaction of the divalent cations with the cell wall prevents access of the antibiotic to the cytoplasmic membrane by mechanisms that remain unclear.