The interactions of the antibiotic lasalocid (LAS) as sodium salt with dipalmitoylphosphatidylcholine (DPPC) multilayers were analyzed in aqueous buffer (pH 7.4) for antibiotic/lipid molar ratios (r) ranging from 0 to 0.12. The study was performed both by X-ray diffraction as a function of temperature (XRDT) and by differential enthalpimetry (DSC). The effect of the antibiotic on the structural arrangement of the phospholipid bilayers is concentration dependent. For 0 less than or equal to r less than or equal to 0.04: (i), the lamellar L-beta phase of DPPC is progressively transformed into a new lamellar phase (called Lx) which displays a higher periodicity. Its composition corresponds to 1 mol of LAS per 25 mole of DPPC (r = 0.04); (ii) no significant structural changes are observed in the L-alpha phase. This finding is important fur biological applications of the antibiotic; (iii) the thermotropic behavior is not markedly affected. Inversely, above r = 0.04, a strong perturbation of the DPPC acyl chain organization is observed which is accompanied by the formation of nonlamellar structures. The disappearance of a lamellar organization for r = 0.08 reflects the destructive effect of lasalocid on the lipidic membrane. The structural and thermal behavior may be explained by two localizations of the LAS molecules in the DPPC multilayers: at r I 0.04, the ionophore molecules as monomers lie parallel to the membrane surface and at r > 0.04, the ionophore as dimers insert in the DPPC bilayer perpendicular to the membrane surface. The change of the orientation of the antibiotic molecules occurs at a specific r corresponding to the composition of the Lx phase. A model for the distribution of the antibiotic molecules in the Lx phase is proposed. A schematic phase diagram is discussed.