SUMMARY. Polymyxin B, an amphiphilic, cationic peptidolipid, which is thought to bind to anionic phosphohpids in cell membranes, is shown to interact with the cellular calcium of cultured neonatal rat myocardial cells in a dose-dependent, partially reversible manner. At concentrations of less than or equal to 0.1 mM, it has two distinct effects. First, it results in displacement of 1.4 ± 0.3 mmol Ca/kg dry weight, which is equivalent to 18.1 ± 3.4% of the total exchangeable cellular calcium. Total calcium displaced by polymyxin B and a nonspecific cationic probe, lanthanum, at its maximal displacing concentration (1 mM), was 5.9 ±1.3 mmol/kg dry weight. Thus, the total displaceable calcium represented 76.3 ± 2.5% of the total exchangeable calcium. Second, polymyxin B (S0.1 mM) causes a reduction in net uptake of calcium, and slows the efflux of both calcium and potassium. Concentrations of polymyxin B higher than 0.1 mM result in an initial displacement of calcium, followed by an irreversible and sustained period of enhanced net calcium uptake. Efflux of calcium is slowed at the higher polymyxin B concentrations, whereas efflux of potassium is enhanced. Cellular contractile activity and electrical activity are irreversibly altered only by the higher concentrations. The results suggest that polymyxin B is a useful probe for the role of membrane phospholipids in control of ion binding and fluxes. (Circ Res 53: 679-687, 1983)