The interaction of negatively charged lipopolysaccharide (LPS) from Pseudomonas aeruginosa (serotype 10) with different kinds of surfaces was investigated by FTIR ATR spectroscopy. The surfaces comprise a hydrophilic germanium plate, a hydrophobic dipalmitoylphosphatidic acid (DPPA) monolayer, a positively charged cross-linked aminopropyltriethoxysilane (ATS) polymer, and a positively charged lipid bilayer consisting of a DPPA inner monolayer attached to the internal reflection element (IRE) and an outer monolayer consisting of equal molar amounts of palmitoyloleoylphosphatidylcholine (POPC) and hexadecylpyridinium (HDPyr) as positively charged component. Whereas the hydrophilic and negatively charged LPS exhibited only weak adsorption to a hydrophilic Ge IRE and to a hydrophobic DPPA monolayer, it bound strongly to the positively charged surface of ATS and interacted also with the POPC/HDPyr membrane. However, in the latter case, the positively charged component (HDPyr) turned out to be not sufficiently strong anchored in the lipid matrix, because it was completely extracted by the endotoxin. The remaining POPC layer exhibited a significantly reduced molecular ordering compared with a compact POPC monolayer. The results demonstrate distinct interactions of the negatively charged LPS with positively charged surfaces and the ability of LPS to remove positively charged molecules from a biomembrane.