The physical structure of the lipid component of the outer membrane of Gram-negative bacteria, here mutants of Salmonella minnesota, was studied with different optical and calorimetric techniques on the bilayer and with film balance measurements on the monolayer system. Special emphasis was laid on the elucidation of the phase behavior of lipopolysaccharides and its isolated lipid component, free lipid A, differing in the length of the polysaccharide moiety. All samples exhibit the phase transition gel-liquid crystalline of the hydrocarbon chains with T c for free lipid A lying well above the growth temperature of the bacteria (37°C)m while the mutant lipopolysaccharides show values in the range 3-–37°C. The state of order of the hydrocarbon chains at 37°C is lowest for the deep rough mutant lipopolysaccharides. Thus, an explanation of the high sensitivity of the mutants Re and Rd against hydrophobic drugs is possible. The behaviour of several physical quantities, e.g. enthalpy and cooperativity of the phase transition indicates that free lipid A and, to a lower extent, also the deep rough mutant lipopolysaccharide (mR595) form inverted structures. From this a model is derived for the mechanism of incorporation of lipopolysaccharide into the outer leaflet of the outer membrane after its lipid and saccharide parts have been translocated separately from the cytoplasmic membrane.