To obtain information on the effects of a divalent cation (Ca2+) on the membrane properties of the lipid A analog, E5531, the particle size, zeta potential, membrane fluidity and pharmacokinctices was investigated in rats after the addition of calcium ion. Within a molar ratio for [Ca2+]/[E5531] <= 3, the presence of Ca2+ increased the zeta potentials of the E5531 membrane, but had no effect on particle size. Ca2+ decreased membrane fluidity and the pharmacokinetics in rats. These effects of Ca2+ on the membrane properties of E5531 particles could be eliminated by the addition of EDTA.