A novel system was used previously to characterize the dynamic interaction of a polysaccharide-deficient, lipid-rich lipopolysaccharide (LPS) with rabbit erythrocytes (RaRBC). Exposure of the RaRBC to the LPS rendered them sensitive to induction of hemolysis by the cationic antibiotic polymyxin B (PB) in a time- and temperature-independent manner. Subsequent decay in the response of LPS-sensitized cells to PB was shown to be critically dependent on both the time and temperature of incubation of RaRBC with LPS and to be independent of a change in LPS binding (Carr and Morrison, Infect. Immun. 43:600-606, 1984). In the present study, we performed experiments designed to define the mechanism by which PB mediates hemolysis of LPS-sensitized RaRBC. Experiments were performed to examine the molecular requirements of the LPS and the PB that were essential for hemolytic activity. The capacity of various cations to mediate hemolysis of LPS-sensitized RaRBC or to block PB-mediated hemolysis and the temperature dependence of the PB lytic reaction were investigated. The results of these experiments suggest that PB-mediated hemolysis of LPS-treated erythrocytes is dependent upon an initial ionic association of PB with erythrocyte membrane-bound LPS, followed by hydrophobic insertion of the PB fatty acid into the erythrocyte membrane lipid bilayer.